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-> The Independent Video Game Community

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    HENkaku Update 3 introduces PSN Spoofing

    Posted Aug 28, 2016 at 6:24 AM by endoverend
    The third update for the PS Vita homebrew platform HENkaku has just been released. Along with the update comes a highly requested feature called PSN Spoofing, which allows users to access the...
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    CEMU 1.5.6 released (Wii U emulator)

    Posted Aug 28, 2016 at 4:24 AM by Costello
    The amazing Wii U emulator (which is already capable of properly playing a good number of games) received an update today with version 1.5.6. The highly technical change log below may not mean much to...
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    Reggie Fils-Aimé explains how Nintendo will not repeat Wii U mistakes for the NX

    Posted Aug 26, 2016 at 7:48 PM by Voxel
    In an exclusive interview with [a]listdaily yesterday, Reggie Fils-Aimé, the CEO of Nintendo of America, explained that Nintendo needs to improve their communication of "the positioning" of the NX...
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    UPDATE: Wololo confirms the potential for Vita piracy

    Posted Aug 26, 2016 at 6:28 AM by Chary
    The wild ride of the Vita scene continues. The well known hacking site Wololo has confirmed that reputable sources have found out that there is a definite way to dump and run backup copies of PS Vita...
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    Civilization VI - 25th Anniversary Edition announced

    Posted Aug 25, 2016 at 6:34 PM by Ericzander
    Civilization Fans rejoice! Earlier today it was announced that the sixth entry in one of the most addicting series since 1991 will come with a 25th Anniversary Edition, especially for the series'...
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    Japanese Distributor JESNET Bought by Nintendo

    Posted Aug 25, 2016 at 6:21 PM by Voxel
    Today, Nintendo said they would buy the company that manages the distribution of their products in Japan, for a total of $46.5 million, and will become a subsidiary of the gaming giant. This will...
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    Sony adds Two-Factor Authentication to PlayStation Network

    Posted Aug 25, 2016 at 6:07 PM by Chary
    After numerous user account compromises and security breaches, Sony has finally added 2FA to the its PlayStation Network. It's highly recommended that users take advantage of this extra layer of...
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    New Nintendo 3DS XL and 2DS colors announced for Europe

    Posted Aug 25, 2016 at 11:29 AM by LuigiXL
    Nintendo of Europe announced that three New 3DS XL color variants will be hitting store shelves on November 11th. The colors are orange/black, white, and pink/white. There will also be a blue/black...

  1. Hatsune Miku: Project Diva X
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Third Bobcat Baseball Professional Update

Third Bobcat Baseball Professional Update

MILLEDGEVILLE, Ga. – This is the third 2010 summer update, checking in on former Georgia College & State University baseball players currently making their way up the professional ranks.

Michael Antonini (2007)

Pitcher for the Buffalo Bisons of the International League, Triple-A affiliate of the New York Mets … Has one start at Buffalo, going six innings in the win, allowing one unearned run and fanning five against Lehigh Valley … Posted a 6-9 record in 23 Double-A starts this season … Had 131 1/3 innings pitched this season, with 106 strikeouts and an ERA of 4.32 … Buffalo currently sits in second place in the International League’s North Division with a record of 64-54.

Brandon Durden (2005)

Pitcher for the Tulsa Drillers of the Texas League, Double-A affiliate of the Colorado Rockies … 4-10 in 19 appearances, 18 of which were games started … Has pitched 106 2/3 innings, good for third on the team … Has an ERA of 5.15 and has struck out 60, good for fifth on the staff … When four innings against the Midland RockHounds Aug. 13, allowing four earned runs in a loss … The Drillers are currently in third place in the Texas League’s North Division with a record of 25-22 in the second half.

Andrew Reichard (2007)

Pitcher for the nearby Augusta GreenJackets of the South Atlantic League, Single-A affiliate of the San Francisco Giants … Is currently 3-0 with a 3.69 ERA … Has struck out 57 batters in 63 1/3 innings of work … Went seven innings for a win Aug. 8, beating the Asheville Tourists by allowing three earned runs and striking out seven … Has thrown at least six innings in his last nine appearances … Augusta is 24-22 in the second half, third in the SAL Southern Division.

Alex Burkard (2010)

Pitcher for the AZL Angels, Rookie ball affiliate of the Los Angeles Angels … Has struck out 21 in 14 2/3 innings of work … Has an ERA of 6.75 … Most recently threw Aug. 8 against the Indians, striking out three while allowing two runs in 1 2/3 innings of relief … The AZL Angels are currently 18-24.

Brooks Robinson (2009)

Infielder for the Kalamazoo Kings of the independent Frontier League … Hitting .230 in 34 games played … Has one home run and one triple … Has scored 13 runs along with 10 RBI … The Kings are currently struggling with a record of 21-54.

Pete Paris (2009)

Catcher for the Windy City Thunderbolts of the independent Frontier League … Has seen action in 18 games, hitting .283 with five RBI and 12 runs … Windy City is 43-31, second in the East Division.

Brandon Anderson (2007)

Outfielder for the Windy City Thunderbolts of the independent Frontier League … Hitting .260 with three home runs and 20 RBI over 27 games … Has also scored 24 runs and stolen 15 bases … Windy City is 43-31, second in the East Division.

Brendon Malkowski (2010)

Pitcher for the Quebec Las Capitales of the independent Can-Am League … Has made four appearances, tossing 10 innings and allowing just one earned run for a 0.90 ERA … The towering righty has also fanned five while walking just one … Quebec is 14-10 and second in Can-Am second half.

The Georgia College Department of Athletics, back-to-back winners of the PBC Commissioner's Cup, sponsors 10 varsity athletic programs at the NCAA Division II level. As a Division II program, Bobcat Athletics prides itself on balancing the life of the student-athlete, evidenced by the teams’ multiple appearances in post-season competition as well as documented academic success and community-service involvement. Sign up at for up-to-the-minute reports, and visit for more information, and to nominate your favorite former Bobcats and Colonials to the GCSU Athletics Hall of Fame.

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GT Featured

The Breakfast Fight Club by Dave MacDowell

Dave MacDowell has to be one of the best pop artists I've seen in a long time. There are so many great works you can find and purchase on his website, so please check it out! My only regret is not finding this earlier in the holiday season so I could gift people!

Newest Posts on GeekTyrant


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Sleeping Dogs ‘Police Investigations’ gameplay walkthrough
posted on 08.02.12 at 02:25 PM EDT by (@salromano)
Wei Shen deals with police affairs.

Square Enix has released a new gameplay walkthrough for Sleeping Dogs. Different from previous clips, where protagonist and undercover cop Wei Shen is shown taking on hits for the Triads, this video shows Shen involved in police affairs such as investigations – some of which are a little too close to home. Watch the clip below.

Save $3 with the coupon code "GEMATSU"
  • bebestorm117

    After this video Im banning myself from watching anymore.. Game is going to be awesome.

    • Zero

      I’m trying to do the same more or less. I watched this one, and it was (once again) awesome.

  • zakou

    This reminded me of watch_dogs

    • whiteferrero

      except somehow, i like this one more. XD

  • Chaos Raiden

    Loving this game, and it is a must buy for me along with Darksiders II after next week.

  • Pirate King

    Looks like it’s going to be even more fun than I predicted!!!

  • KingOptimusOrigins111

    Still looks okay.


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Someone wrote…

I think I need to realise that people aren’t as open-minded as me sometimes. Like, I’m an asexual who is romantically attracted to masculinity but masculine girls are still on the cards. They still fall under masculinity, right?

What’s your experience?

And what are you thinking about gender right now?

Posted by on September 27th, 2013 at 08:00 am

Category: your voice One comment »

One Response to “Attraction”

  1. Callista

    I feel ya! Being asexual makes it hard enough to find potential dates without people going, “Uhh… but I’m a chick! You can’t possibly be attracted to me if you’re attracted to the masculine!” They seem never to have thought about the fact that being female doesn’t restrict your access to masculine traits.

    I’m ace too, and the things I’m attracted to are entirely independent of gender–compassionate, intelligent, unique people. It’s an awkward position to be in–it’s like I’m pansexual, only without the -sexual. I don’t fit in anywhere, really.

    I have to remind myself, it’s okay not to fit in anywhere.

Leave a Reply

Can I show your picture? If you have a Gravatar associated with this email address, it will be displayed as your photo. If not, I'll just put a picture of a fork next to your comment. Everybody likes forks.

Be nice. Judgmental comments will be quietly deleted and blacklisted. There's plenty of room for those elsewhere on the web.

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text:
Monday, January 2, 2012

Mean Spirited?

I had a very interesting and thought provoking comment to my post about my top ten worst practices in genealogy. I certainly apologize to anyone who was offended by the post. But I might say that offense was and is predictable.

I can remember one experience I had when I was helping a person with Personal Ancestral File. The person was having a great deal of trouble understanding how to enter information into the fields. I had spent a considerable time trying to help short of taking over and doing the work myself. After making one suggestion about how to enter the information, the person turned to me and said, essentially, "I want to do it my way, not your way, I don't want to know anymore." Is it mean spirited to simply decide that you are not longer welcome to help? My comments concerning the different problems I encounter are and were, as I clearly said, specifically given for didactic purposes.

I recently had an erstwhile genealogist tell me that he refused to learn computers and would not listen to me try to help him. He said he was a mechanic and had quit his job when he had to learn about computers to fix cars.  Was it mean spirited to take him at his word about not wanting to learn how to do computers for helping with his genealogy?

The commentator is right in many ways, one of which is that I am not well suited to help with certain types of genealogical problems especially when the person in question does not want to learn and will not listen to an explanation at any level. I have dealt with people who come into the FHC with the same problems day after day, until they have talked to every single volunteer more than once and have never either listened to what was told to them or changed in any way.

One time I was helping a patron with his "brickwall" problem in North Carolina. He carefully explained to me that he and his family had been searching records for years without results. I asked him where the person lived and when they were supposed to live there? He had a name, a date and a place. I suggested looking to see what county the town was in at the time in question. He did not know the county. I looked in the Redbook, (Eichholz, Alice. Redbook: American State, County & Town Sources. Provo, Utah: Ancestry, 2004. ) to see what the county was at the time in question and told him that the records were likely in several different counties depending on the time period. He began to argue with me, saying he did not need to look in those counties because he already knew where the town was located. This discussion went on for some time while I tried to help him understand that the county boundaries had changed and that the records he was looking for might be in any of four or five different counties. He simply refused to listen to anything I had to say on the subject and went away without bothering to write down the counties or even to look at the resources we had in the library. Some people cannot be helped no matter how patient or helpful you wish to be.

Some of the problems I encounter are not limited to those who are not "experts" and some problems resist both education and patience. How do you approach a person who sincerely believes that they have traced their ancestry back to Adam? I am not asking this question in a sarcastic or mean spirited way. Some people have specific background beliefs that, as a genealogist, I am not going to address. The issue of whether or not accepting books like Burke's Peerage and Gentry is one thing, but extending your ancestral lines through a traditional connection to Biblical passages is not genealogy as I define it. Strictly speaking, this is not a genealogical problem at all, but a cultural and historical one.

The commentator used an example of helping a child learn to walk. None of the issues I set forth in my post are really comparable to helping a child to learn to walk. I don't get the connection between learning to walk and tracing your genealogy back to Adam.

When I was operating a retail store, we used to have customers who came into the store and started out by asking, if the price we had on the products was our lowest price? Would we negotiate the price? Have you tried this recently at your local grocery store? Try going into the store and asking if the price they have for milk that day is negotiable? Can I predict the reaction from the store manager? Try going into an AT&T store and try negotiating the price of an iPhone.

Genealogists are the most inclusive, patient, kind and helpful community of which I am aware. But there are really a certain class of challenges peculiar to genealogy that are more or less, beyond help. As I repeat, some of these problems are addressable by education, some are not. They are all commonly encountered by the average genealogist trying to conscientiously do their genealogy and help others.

  1. Let me just say that I enjoyed your initial post and identified with many of the "worst practices" you identified. I don't think you need to apologize in any way. I enjoyed reading your clarification in this post, too. You are right that some people just can't be helped.

  2. Those are probably the same people who repeatedly ask their pharmacist "What can I take?" instead of "How can I prevent?"


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Gene Quintana

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About Gene Quintana
Datsolalee Miniatures Collection

A very large and nice Siletz basket with decorated cover
Item#: 1285N
A very large and nice Siletz basket with decorated cover
The basket is decorated on the sides and cover with arrowheads. On the sides are two leather handles (one of which is loose).
Circa: 1900
Diameter: 14 1/4in
Height: 19 3/4in
Price on Request
An enormous Siletz basket
Item#: 8116
An enormous Siletz basket
Western Oregon
Provenance: The Green Collection. Sold at Sotherby Parke-Bernet May 1972 #28
Circa: 1910
Diameter: 28in
Height: 28in
Price on Request

For Pricing, Contact Gene Quintana via phone or email (below).

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Fresh Like Us

Chiddy Bang

Fresh Like Us Lyrics

[Verse 1]
Now let’s see, the boy’s so zesty, I’m fresh come get me
You don’t gotta know how I does it the best
And this is the guideline on how to keep fresh

Flow blue streak like Tulley, and I’m so gully
Rhyme so cold that it came with a skully
And them haters really love me
Cause they talk always seem to seep out like soda when it’s bubbly
Yea, I’m up there with a small arrow
Oh they mad cause I Shyne like Jamal Barrow?
And they say I’m so proud, but I’m like, I know
I do this thing nobly like Manu
Ten I know, grand like piano
Stack in my pocket, I could make it to the condo

I could take her and U2 like Bono
They say you got a point like Rajon Rondo

One, two, three, if you tryna’ get fresh like me
One, two, three, if you tryna’ get fresh like me
(Fresh like this, are you fresh like that?)
One, two, three, if you tryna’ get fresh like we
(Fresh like this, are you fresh like that?)
One, two, three, if you tryna’ get fresh like we

[Verse 2]
Now I’m fresh like that
I’m the best like that
I’m Aretha on your speakers so respect like that
I see them ladies in the house, they better than cold weather
So naturally my number I slip her like chancletas
And I’m the one that they came to see
Ayo, my drink keep it mixed like Amerie
I be grindin’ like a dancer, flow rarer than pandas
School of hard knocks, no I will never transfer
Money on top, my money on top
When she give me brain it’s a dummy on top
Regret if I let her take all my guap
She funny like a episode of the Boondocks
You watch, I get my point across like Swatch
I’m on another boat, don’t cross my yacht
The sonic is chronic, I know I can’t stop
I guess I got nicks like Dr. Robot

[Verse 3]
It’s time to throw a party
Hey, like my girls French, like Louie V and Cartier

Make the people jump, you could pass the Courvoisier
You gonna’ need a couple more shots, to get on my level
Hit on the pedal, gas up a female
Oh, they made cause I’m smooth like CL?
You don’t know, I’m a big deal
Stand up like Hughley, I keep it on the D.L

Yea, I’m inbox like G-mail
I get head, I need tail
But let me quit it ma’, no play
I was tryna’ break in like O.J
Okay, I be puffin on a Starbury
So if I interview, I’m acting like Marbury
They tend to get aggie
But if hate is the new love, then we fittin’ to get married

Fresh like this, are you fresh like that? (x6)

About “Fresh Like Us”

A guidline on how to keep fresh, according to Chiddy Bang. The song Ce Jeu by Yelle was sampled for the beat. The lyrics of this song feature Proto’s signature similies (notice how many times the word “like” is used)

Track Info

Recorded At

Release Date January 1, 2008

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Element of Surprise

La the Darkman

Featuring Masta Killa, U-God

Produced By 4th Disciple

Album Heist of the Century

Element of Surprise Lyrics

[La the Darkman]

Bear witness to the god, young thugs don't live long
Life is a game of chess (and you playin the pawn)
I be the king, listen to the knowledge I born
Walk a righteous path, you can never go wrong

Yo east New York, Schenectady, this kid live next to me
Manifest his destiny, to specialize in treachery
Subliminal, wanted to be nuttin, but a criminal
Wit braids, and doo-loo dropped out the eight grade
Tradin rocks, mad obsessed wit guns, was infected
Should of came amongst gods, could of been resurrected
He expected to live long, holdin the Glock
Never opened up his lessons, never took the salat
Robbed a liquor store, the old man knew him since four
Still slapped him wit the toast, and son emptied the cash drawer
Watchin news flicks, what the kid did, gettin famous
Found the owner in the back of the store, left brainless
The stainless was found, in the sewer three blocks down
The DE's flashed his picture around the Polo Grounds
Kicked his grandmother door down, wit a search warrant
Hand cuffed him in his boxers, tipped off by an informant
He was sentenced to thirty-two, Y Equality
Allah rules
, he should of listened to his jewels

Chorus 2x
[La the Darkman and Masta Killa]
Young thugs don't live long
Life is a game of chess, and you playin a pawn
I be king, listen to the knowledge I born
Walk a righteous path, and you can never go wrong (word up)

[Masta Killa]

This be the dance of the drunken, nigga found slunken
Over the steering wheel with one to his head, black
All those he drove his next destination of home
Suddenly sniped from civilization
Reality starts to set in, his last thoughts
Flashin back to where it all begun
A shorty who slung for a militant soldier
Who made his bones by holdin down the corner
Never turned snitch even though he was pinched by the Feds
He held his weight and did a bid
Of two to six, as he sat from his cell wit a vengence
He held in his heart like a icepick
Physically, he's cut the fuck up
Tryin to maintain but his head is fucked up
To learn, the word on the streets confirmed
Of the sickness, murderin a family member that turn
Informant, he swore to a code of silence
He broke it, this lead to bloodshed and violence

This chick Iris out in Queens had a cocaine Lexus
Dime sexes, she rock a leather G necklace
Building reckless, Queen kingpin deathwish
Hit women that'll fuck you and return them the essence
This evil widow, she pulled a fo'-fo' out the pillow
Wit a silencer, la costra nostra cats, they admired her
They hide a force sting to hit the North Region king of teamster
Head C.E.O. of, a major ring but one thing
Lead to another, a leaf, blew her cover
She got side-swiped, tied up, he buck fifty cut her
He shot her both knees then, dumper her in the gutter
It was gory, the top nigga tortured her for glory
But, he let her live so she could about this story

About “Element of Surprise”

Track Info

Recorded At

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text:
Sunday, June 24, 2012

a perfect day

On the porch, shortly after 7 a.m., outside the door which is ajar, the day is perfect. Cloudless sky, bright sun, a single mourning dove crooning. The sun reaches in and dances on a forearm with the morning cool ... warmcool, coolwarm. Everything is so flawless. It is almost ridiculous, as if an inexperienced high school drama class had staged it all.

But perfection positively demands some imperfection and so I waited patiently for the body to reassert the aches and pains of my age and wondered what thought-ghosts would request attention and solution. It was early yet. I had not had quite enough coffee and so, for a little, the perfection of the day had its uninterrupted say: There is no forgiveness; there is just forgiving ... always.

And then the first of the thought-ghosts tip-toed in, a bit groggy still, but assured of its place in the pantheon of imperfections I would soon insist on: "He's so dumb, he would fuck up a wet dream."

Later, I will go out and do a little zazen. I will leave the door open on this perfect day and the air and light will pour in without a backward glance. It's the beginning of spider season, and here and there in the zendo, critters only slightly less gossamer than their webs will move about, taking care of business. In my imagination, they will forgive me the incense, the candle, the sounding of a small gong, but of course they really have no forgiveness to offer. They are forgiveness like the day. Always forgiveness... like the day ... every day.

Yesterday, in the supermarket, I found myself standing next to an energetically thin woman who was loading her grocery cart with super-saver packages of chicken thighs. She had five or six in her carriage and was reaching for more. "You're really going to feed the troops tonight," I commented idly. She swung into the banter seamlessly, explaining that the chicken was for her dogs. Her face was bright and open and it was a pleasure to listen to someone who just liked what she liked and did what she did. She disabused me of the notion that dogs shouldn't be given chicken ... "that's just the cooked chicken. Raw chicken is OK. And it saves on veterinarian bills. I have never found a bone in their droppings." And when she priced it out, chicken was in about the same price range as prepackaged dog food. Since she lived some 40 minutes away, up in the hills, she was loading up on the thighs, which were on sale.

Bright and friendly as a spider that gives no thought to bright or friendly or the forgiveness that is the day.

A Zen Buddhist monk I have known through email once told me that a Korean monk friend had suggested that perhaps, "suffering is just the resistance to pain."

And as the aches and pains rise up and as the ghost-thoughts heave themselves out of bed, I wonder if it is not also true, "suffering is just the resistance to forgiveness." Not the usual smarmy "forgiveness," but the forgiveness that cannot be escaped, the forgiveness of spiders, the forgiveness of coolwarm and warmcool.

Dumb enough to fuck up a wet dream. Well, wet dreams cannot help their forgiving nature any more than a spider or a mourning dove can. In the brightness of the sun and the perfection of the morning air, who could forgive or be forgiven. To forgive and be forgiven is a wet dream waiting to be fucked up.

Perfectly fucked-up. Don't be shy.

Wet dreams don't mind.

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text:
Open Access

Accurate proteome-wide protein quantification from high-resolution 15N mass spectra

  • Zia Khan1, 2, 3,
  • Sasan Amini2, 4, 5,
  • Joshua S Bloom2, 4,
  • Cristian Ruse6,
  • Amy A Caudy2, 7,
  • Leonid Kruglyak2, 8, 9,
  • Mona Singh1, 2,
  • David H Perlman2, 4, 10 and
  • Saeed Tavazoie2, 4, 11Email author
Contributed equally
Genome Biology201112:R122

DOI: 10.1186/gb-2011-12-12-r122

Received: 11 July 2011

Accepted: 19 December 2011

Published: 19 December 2011

In quantitative mass spectrometry-based proteomics, the metabolic incorporation of a single source of 15N-labeled nitrogen has many advantages over using stable isotope-labeled amino acids. However, the lack of a robust computational framework for analyzing the resulting spectra has impeded wide use of this approach. We have addressed this challenge by introducing a new computational methodology for analyzing 15N spectra in which quantification is integrated with identification. Application of this method to an Escherichia coli growth transition reveals significant improvement in quantification accuracy over previous methods.

Experimental methods for proteome-wide quantification using liquid chromatography coupled mass spectrometry (LC-MS) rest heavily on computational methods that analyze mass spectra for peptide and protein quantification [1]. Computational analysis typically relies on two main components: (1) a peptide identification algorithm that assigns amino acid sequences to fragmentation spectra by searching a database of theoretical peptides obtained by an in silico digest of an organism's proteome; and (2) a quantification algorithm that collects peaks across mass spectra in the form of extracted ion chromatograms (XICs) over the duration of chromatographic elution of peptides.

These algorithms have been shown to produce robust and accurate peptide quantification results across a wide range of quantification strategies. They are an important part of the data analysis employed in a widely used experimental approach called stable isotope labeling by amino acids in cell culture (SILAC) [2, 3]. In most typical applications of this approach, a sample in which stable isotope-labeled amino acids are incorporated metabolically by an organism is compared to an unlabeled sample from a different experimental condition. Because both samples are combined prior to protein extraction, the samples are subjected to the same extraction, sample handling, digestion, chromatography, and ionization conditions. This eliminates much of the technical variation between individual samples. Consequently, the final relative abundance measurements, in the form of ratios of the areas of paired XICs from both the unlabeled and labeled peptides, are highly accurate. To ensure that only labeled amino acids are incorporated into proteins, amino acid auxotrophs are typically employed in conjunction with labeled amino acids, rendering this strategy unavailable for prototrophic microorganisms, such as wild-type bacteria and yeast.

15N-labeling through the metabolic incorporation of a single source of labeled nitrogen provides an alternative strategy for labeling virtually all nitrogen in expressed proteins of prototrophs and auxotrophs [4]. 15N-labeling has three notable advantages over labeling with specific amino acids. First, all peptides are labeled, irrespective of their amino acid composition, and therefore any endogenous peptide or peptides produced by any chosen endoprotease can be used for quantification. The use of multiple endoproteases has been shown to significantly increase the number of distinct, non-overlapping peptides identified per protein [5]. Second, the label is not subject to the complications of metabolic conversion of amino acids [6]. Such label conversion is a particular problem in yeast, where arginine is efficiently converted into proline [7]. Third, labeled ammonium salts are much less expensive than labeled amino acids and are efficiently used as a nitrogen source by many microorganisms, including Escherichia coli and both budding and fission yeasts.

Despite these advantages, labeled amino acids have been used in favor of 15N-labeling for proteome-wide quantification in microbes [8]. Amino acid labels typically produce a small number of distinct mass differences between pairs of XICs, allowing all XICs and XIC pairs to be found prior to database search. In contrast, 15N-labels produce mass differences that depend on the length and composition of a peptide, complicating the detection of such pairs. Existing methods for 15N quantification rely on peptide database search of fragmentation spectra to locate paired peaks in parent mass spectra [6, 912]. Statistically significant matches to a merged database of both unlabeled and 15N-labeled peptides provide the labeling status of a fragmentation spectrum and an amino acid sequence from which nitrogen composition can be determined. During quantification, these methods find peaks within parent mass spectra that fall within an interval centered at the precursor retention time and precursor mass-to-charge ratio (m/z) of the peptide-assigned fragmentation spectrum. Then, a mass difference, computed using the nitrogen composition and precursor charge, is used to find peaks in parent mass spectra that correspond to the XIC with the opposite labeling status. By examining only peaks in parent mass spectra near a fragmentation spectrum and at the expected mass difference, existing methods use only a small fraction of the information in all the parent mass spectra to distinguish signal from noise, resulting in noisy peptide quantifications. Therefore, the lack of a robust quantification method significantly limits the wide use of 15N-labeling for proteome-wide quantification in prototrophic microbes.

We have developed a novel approach for 15N quantification that finds all XICs as a first step, reliably using all the available information in all the parent mass spectra. Once XICs are found, our method pairs these XICs by using two databases of labeled and unlabeled intact peptide fragment masses and peptide nitrogen counts. Then, pair membership allows the method to determine the label status of a fragmentation spectrum and search the appropriate peptide database. Additionally, the XIC pair provides a constraint on the nitrogen composition, allowing the search to limit the peptides scored against any given fragmentation spectrum. In an experiment to compare the proteome of stationary phase E. coli with that of exponentially growing cells, we show that our method significantly improves the quantitative accuracy of protein abundance ratios obtained from 15N mass spectra.

Results and discussion

Our method finds XICs prior to database search of fragmentation spectra using techniques we have previously developed [13]. From the accurate mass and charge information assigned to a monoisotopic XIC, our method searches entries in unlabeled and 15N peptide databases and uses the returned nitrogen counts to examine a limited number of 15N mass differences (Figure 1a). After unambiguously pairing XICs, the method designates each XIC as originating from either the unlabeled or the 15N-labeled sample. This process is repeated for all monoisotopic XICs until no more XICs can be paired.
Figure 1

Approach for peptide quantification using 15 N mass spectra. (a) The method uses accurate mass and charge measurements to search a 14N-unlabeled database and a 15N database of intact peptide masses and nitrogen counts obtained by in silico digestion of an organism's proteome. The number of nitrogens in each of the returned peptides is used to examine a limited number of mass differences designated by arrows. On finding an unambiguous 14N-15N pair, the method labels each member of the pair as originating from either the unlabeled or the 15N-labeled sample. (b) During peptide identification, a fragmentation spectrum (1) associated with a member of the pair is searched against an unlabeled database or 15N-labeled database of peptides based on the assigned label status. Note that only one member of the peptide pair needs to have an associated fragmentation spectrum. The monoisotopic masses of the peptides (2) and their nitrogen composition (3) are used to limit the search space of peptides scored against the spectrum. The intensity of each member of a 14N-15N pair is used to derive a peptide ratio. (c) Top, a population of incompletely labeled peptides can generate a complex isotope distribution pattern for 15N-labeled peptides. The peaks in purple, in order of decreasing m/z, correspond to peptides lacking one and two labeled nitrogens. Bottom, even in highly enriched samples, such peaks can be found in real mass spectra as illustrated for a +2 charge peptide, LGFFETVDTDTQR from E. coli protein yaeT (b0177). (d) For a range of charge states, monoisotopic XICs can be paired with an XIC with an additional 13C, but lack any XICs detected at a negative 13C shift (top). In contrast, 15N XICs will lack both XICs at negative and positive 13C shifts (bottom).

During peptide identification, the method iterates through paired XICs that have an associated fragmentation spectrum. Based on the label assigned to an XIC, our method searches the fragmentation spectrum against an unlabeled database or a database of 15N-labeled peptides. In addition to the mass of the peptide, this search uses the discovered constraint on the nitrogen composition of a peptide from the pairing phase to limit the search space of peptides scored for identification (Figure 1b). Last, the peptide expression ratios are obtained from the areas of paired XICs. Remaining, unpaired monoisotopic XICs that have associated fragmentation spectra are assigned a labeling status based on the highest scoring search result from the unlabeled or the labeled database.

A critical element of our approach is the identification of monoisotopic XICs. This process is complicated by the presence of incompletely labeled peptides. Even in highly enriched 15N-labeled samples, a population of incompletely labeled peptides can generate a set of XICs that have a more complex isotope distribution pattern than the unlabeled sample (Figure 1c). To distinguish the monoisotopic XIC from this incompletely labeled population, we leverage the precision of modern high-resolution mass spectrometers. Our approach rests on the observation that the difference d = 0.00631994439 Da that exists between the 0.99703489341 Da shift between 15N isotopes and the 1.00335483781 Da shift between 13C isotopes for charge +1 peptides allows these incompletely labeled XICs to be distinguished from 13C XICs. If the precision p of a high-resolution instrument approaches 1 part per million (ppm), this shift can be distinguished at up to (d / p) × 106 = 6,319 in m/z, above the maximum measured in a typical bottom-up proteomics experiment. At a charge state of +4, this shift can be distinguished at up to 1,579 in m/z. Examples of such shifts can be found in real spectra, as illustrated for a pair of peaks generated by unlabeled and labeled populations of a +2 charge peptide, LGFFETVDTDTQR from E. coli protein yaeT (b0177; Figure 1c, bottom). Here, the error between the theoretical shift and the observed shift between the peaks generated by the population of peptides lacking a single 15N and the fully labeled population is 4.4 ppm and 0.3 ppm for 13C and 15N, respectively. An instrument with precision of 4 ppm would be sufficient to identify this shift as originating from a 15N isotope, as opposed to a 13C isotope. To leverage the precision of modern high-resolution instruments, our method labels XICs as monoisotopic if, for a range of charge states, no XIC is found at a negative 13C shift and an XIC is found at a positive 13C shift (Figure 1d, top). An XIC is labeled as undetermined or 15N if no XICs are found at both negative and positive 13C shifts for a range of charge states (Figure 1d, bottom). Additionally, if a 15N or undetermined XIC was selected for fragmentation, its associated fragmentation spectrum can also be transferred to the nearest monoisotopic XIC at a positive 15N shift to increase identification rates [14]. Also, in experimental samples where high enrichment levels cannot be obtained, due to limitations in labeling efficiency and impurities in the nitrogen source, the intensity information in 13C and 15N XICs found by our high-precision approach can be leveraged to generate ratios that are adjusted for significant populations of these partially labeled peptides [14].

In order to evaluate our approach, we performed an experiment to quantitatively compare the proteomes of stationary and exponential phase E. coli (see Materials and methods). We selected these two conditions because the transition to stationary phase is well-characterized, and the expression levels of a large number of proteins during this transition are known to differ over a wide range [15]. Stationary and exponential phase cells were harvested from 15N-labeled and unlabeled media, respectively. Cell lysate was prepared, combined, and rendered into peptides by trypsin digestion, and then subjected to two-dimensional nano-flow LC-MS analysis.

We analyzed the resulting MS data, comparing our approach to the method employed by XPRESS and Mascot Distiller v.2.3.2. Both methods rely on finding XIC pairs by searching fragmentation spectra against a merged database of both unlabeled and 15N-labeled peptides [10, 16]. The parameters for XPRESS and Distiller were selected to be appropriate for the precision and mass accuracy of the data. In order to evaluate the methods, we examined the consistency among ratios measured from distinct peptides belonging to the same protein. We expected that distinct peptides from the same protein would be determined to have approximately the same ratios of stationary phase to exponential measurements. For proteins with two or more distinct quantified peptides, we randomly assigned the peptides from each protein into one of two groups and computed the median log2 ratio measured for peptides in the two groups. The two ratios, from peptide group 1 and peptide group 2, were correlated for XPRESS and Distiller as well as our method (Figure 2). The significantly higher Spearman's correlation for our method (0.96 our method, 0.84 for XPRESS, and 0.67 for Distiller) indicated that our method produced significantly better quantitative measurements and peptide identifications.
Figure 2

Comparison with XPRESS and Mascot Distiller. (a-c) Correlation of log2 ratios between distinct peptides from the same protein obtained by Mascot Distiller (a) [16], XPRESS (b) [10], and our method (c). Peptides from each protein were randomly assigned to two groups for this comparison of log2 ratios of peptides in group 1 versus log2 ratios of peptides in group 2. Multiple measurements (for example, charge states) of the same peptide, when present, were collated and given a median log2 ratio prior to forming the peptide groups. Black points designate proteins identified and quantified by both methods, and red points designate proteins identified and quantified that were specific to each method. The diagonal gray line designates perfect correlation.

Both Distiller and XPRESS reported more proteins (N = 664 Distiller; N = 554 XPRESS) with two or more quantified peptides than our method (N = 424). This difference in total number of quantified proteins reported by the methods could either be due to differences in sensitivity of accurately determined proteins, or differences in accuracy of quantification. To investigate this, we observed that the Spearman's correlation for proteins (N = 385 Distiller; N = 373 XPRESS) quantified by two or more peptides by our method and each of Distiller and XPRESS independently was higher for our approach (0.98 our method; 0.79 Distiller; 0.89 XPRESS). Next, we examined the correlation for proteins specific to XPRESS (N = 181), Distiller (N = 226), and to our method (N = 51). For these proteins, we computed significantly higher correlations for our method (0.87 our method; 0.49 Distiller; 0.65 XPRESS), suggesting that erroneous pairing of unrelated peaks and subsequent mis-quantification of the assigned peptides contribute to the inferior performance of XPRESS and Distiller.

We additionally examined the raw mass spectra corresponding to the XICs of the one outlier protein yihX (b3385) reported by our method (Figure 2c). The outlier was a peptide with the sequence VLGAWSDLTR. From parent mass spectra, it appeared that an unknown peptide with nearly the same mass co-eluted with both the labeled and unlabeled versions of VLGAWSDLTR, leading to incorrect detection of XICs. However, the exact cause of the problem was difficult to discern by eye since the peaks belonging to the XICs were of very low intensity.

In order to evaluate the reproducibility of our method, we conducted a label-swap experiment. We independently cultured two identical sets of stationary phase and exponential phase cells differing only by which culture was grown in the 15N labeled media. Analysis of both biological replicates by our method enabled quantification of 539 proteins and 1,985 peptides at a stringent 1% peptide-level false discovery rate (FDR). High correlations across protein and peptide ratios (Spearman's 0.96 and 0.94, respectively) from the two biological label-swap replicates indicated that the analysis was highly reproducible (Figure S1a,b in Additional file 1).

The label swap experiment allowed us to investigate the apparent increase in variance from XPRESS for negative log2 peptide ratios (Figure 2b). When we conducted the same analysis using XPRESS, we found the reverse pattern of increasing variance towards positive log2 ratios (Figure S2a in Additional file 1). No such increasing variance was observed from our method (Figure S2b in Additional file 1). These results indicate the increasing variance in the negative range in XPRESS is linked to the growth condition of the cells and may reflect that XPRESS is sensitive to the differing expression levels of proteins in the two experimental conditions. Additionally, our method reported slightly more (N = 408) proteins with two or more quantified peptides than XPRESS (N = 399) for the label-swap. Correlations across peptide ratios were higher for our method overall (0.97 our method; 0.84 XPRESS), for proteins in common (0.97 our method; 0.85 XPRESS), and proteins specific to each method (0.94 our method; 0.79 XPRESS). This additional result provides further evidence that indicates our method provides more accurate quantitative measurements and peptide identifications than XPRESS.

The label-swap experiment also allowed us to examine the number of multiply coupled monoisotopic XICs and unpaired monoisotopic XICs present in 15N mass spectra: 221 monoisotopic XICs in the forward experiment and 254 monoisotopic XICs in the label-swap experiment were multiply coupled. For comparison, 5,556 monoisotopic XIC pairs were found in the forward experiment and 5,224 monoisotopic XIC pairs were found in the label-swap experiment. Unpaired monoisotopic XICs could be generated by several experimental factors: a missed pairing, a multiply coupled XIC that was left unpaired, a protein that is expressed in only one of the conditions, an expression difference outside the detectable range, or contaminant compounds. As all of these factors contribute unpaired XICs, we found in the forward experiment 28,426 monoisotopic XICs of which 17,093 were left unpaired and in the label-swap experiment 31,256 monoisotopic XICs of which 20,554 were left unpaired.

We additionally used the identified XIC pairs from the label-swap experiment to estimate the precision of our data set. Using the nitrogen composition of the peptide sequence assigned to a pair, we calculated an expected theoretical shift between the two XICs. By computing the difference between the measured shift between the XICs and the expected theoretical shift, we created a histogram of precision measurements in parts per million. The mean precision was 0.151 ppm and the standard deviation was 0.987 (Figure S3 in Additional file 1). Our estimate of precision indicates that the difference between 15N isotopes and 13C isotopes can be detected for charge states +1 and +2 for about 90% of the XICs for the entire m/z range measured. For peptides with charge states +3 and +4, this m/z limit reaches approximately 1,301 and 976, respectively.

In order to deepen our quantitative analysis of the proteomes to include proteins of lower abundance, we subjected our original protein sample to additional fractionation steps. First, the sample was partitioned based on protein solubility into two fractions: high solubility and low solubility. Next, peptides derived from each of these two protein-level fractions were further fractionated using strong cation exchange (SCX) chromatography (see Materials and methods). These additional fractionation steps enabled quantification of 2,320 distinct proteins, 14,605 distinct peptides, and 29,658 quantified XIC pairs at a 1% peptide-level FDR (Additional file 2 and Materials and methods).

To evaluate our method on this deeper survey of the proteome, we examined the correlations between the ratios measured for different peptides from the same protein (Figure S4a in Additional file 1). High correlations (Spearman's 0.96) indicate that our method could accurately identify and quantify distinct peptides from the same protein to measure the fold-change of the whole protein. In addition, we correlated ratios between charge +2 and charge greater than +2 isoforms of the same peptide (Figure S4b in Additional file 1). Although the differing charge states produce very different fragmentation spectra, the peptide ratios from the two charge states were highly correlated (Spearman's 0.98), confirming the high degree of accuracy in both our quantifications and our assignments for each peptide. Next, we examined the intra-experiment variability of the ratios reported by the method. From the ratios of distinct peptides from each protein, we calculated a protein level median coefficient of variation (CV) of 18.4% (Figure S5a in Additional file 1). We also calculated a peptide level median CV of 11.9% from ratios of peptides observed in multiple fractions or with differing charge states (Figure S5b in Additional file 1). These low CVs at both the protein level and peptide level indicate high quantification accuracy. In summary, the depth and accuracy of this analysis illustrates the utility of our method for large-scale proteomics studies.

We further used the data from the additional fractions to evaluate the sensitivity and specificity gained by using the discovered constraint on the nitrogen composition of a peptide from the XIC pairing phase to limit the search space of peptides scored for tandem mass spectrometry (MS/MS) identification. We independently analyzed each of the 24 fractions in two ways: with our method's nitrogen composition constraint imposed and with it deactivated (Additional file 3). When our method was applied to the data, the MS/MS score cutoff required to maintain a constant 1% FDR was significantly less for all charge states, indicating statistically significant gain in sensitivity (paired t-test, P < 3.69 × 10-10 for charge states +1 and +2 and P < 1.35 × 10-11 for charge states +3 or greater). When our method was deactivated, the highest scoring search results above the 1% FDR threshold were free to have nitrogen compositions that did not match the composition discovered by XIC pairing. As these are likely incorrect peptide identifications, they can be removed by simple filtering. After filtering, the total number of search results and quantifications returned by our method on the same data was significantly higher (Wilcoxon signed rank, P < 9.68 × 10-6). This additional result indicates that our method provides a statistically significant gain in specificity.

Next, we investigated whether the quantitative output of our method captured expression level changes of proteins known to differ between exponential and stationary phase. Globally decreased rates of translation and ribosome biosynthesis characterize stationary phase growth [17]. In strong agreement with expectation, we observed that 49 of 51 quantified ribosomal proteins were down-regulated by a similar amount upon transition into stationary phase (Figure 3a). Another known feature of stationary phase is that cells adapt to higher cell density and nutrient limitation by inducing the expression of many stress proteins, including starvation-induced DNA protection protein, Dps, and osmotically inducible proteins, OsmC and OsmY [15]. This is orchestrated by several regulators, including the RNA polymerase sigma factor, RpoS [18]. We observed elevated expression levels of each of these proteins in stationary phase cells (Additional file 2), in agreement with previous results.
Figure 3

Ribosomal protein co-regulation and functional enrichment analysis of protein expression ratios. (a) Box and whisker plot of the log2 ratios of stationary over exponential phase for 51 ribosomal proteins from the low solubility protein fraction. Boxes designate the quartiles. The thick centerline designates the median. Thin lines illustrate the range. (b) A graphical representation of the functional enrichment analysis method from [19] applied to protein expression ratios comparing stationary to exponential phase of growth in E. coli computed by our method. In this representation, rows correspond to significantly informative pathways and columns correspond to 10 equally populated expression bins of log2 ratios (stationary over exponential). Colors indicate pathway over or under-representation levels across the expression bins. Red designates (in log10) over-representation of a particular pathway in any expression bin, whereas, blue designates under-representation.

Finally, to examine whether our measured protein expression differences on a global level accurately characterize the differences between exponential phase and stationary phase cells, we conducted a functional enrichment analysis that bins expression differences based on their magnitudes and directions and determines which biological processes are over- or under-represented in these bins (Figure 3b) [19]. Our results demonstrated that, in addition to cessation of ribosome biosynthesis [17], we captured many other adaptive physiological changes across major biological processes that are known features of stationary phase in E. coli. These include repression of flagellum biosynthesis and motility associated processes [20] and down-regulation of proteins involved in oxidative metabolism [21].

We have introduced a new computational analysis method that significantly improves the accuracy of proteome-wide protein quantification from 15N mass spectra. The method combines two ideas, one to use pairing information to constrain database search, earlier proposed in [2], and another to pair XICs using nitrogen counts, earlier proposed in [22], to obtain an integrated approach for identification and quantification. Using protein samples from a well-characterized growth transition in E. coli, we show that the new method enables the collection of high-quality quantitative data for large-scale proteomics experiments, which is validated by numerous metrics, including its consistency with previously published observations. More broadly, the method allows researchers in the fields such as microbiology to leverage the robustness and accuracy of metabolic labeling without the experimental limitations and complications of amino acid labeling approaches. Because of this practical advantage, we anticipate that our method will provide a valuable tool for proteome-wide protein quantification.

Materials and methods

Strain and media

E. coli K-12 MG1655 was used for all bacterial experiments in this work. Bacterial cells were grown in M9 minimal media with 0.4% w/v glucose as carbon source. The M9 media contained 12.8 g Na2HPO4-7H2O, 3 g KH2PO4, 0.5 g NaCl, 11 mg CaCl2, 241 mg MgSO4, and 1 g NH4Cl (with either 14N or 15N) per liter. 15N-ammonium chloride was purchased from Cambridge Isotope Laboratories (Andover, MA, USA). Growth conditions: overnight cultures of E. coli MG1655 were diluted into fresh M9 media (1:500) and shaken at 250 rpm at 37°C. Cultures were harvested at OD600 values of 0.2 and 0.9 for exponential and early stationary phase samples, respectively.

Protein extraction

We harvested 100 ml of exponentially growing cells (in 14N media) and 22 ml of stationary phase cells (in 15N media) by centrifugation and these were frozen in an ethanol-dry ice bath. The pellets were re-suspended in 1 ml of B-PER bacterial protein extraction buffer (Pierce, Rockford, IL, USA), pooled together, and vortexed vigorously for 1 min. The mixture was centrifuged at 13,000 rpm for 5 min. The supernatant (high solubility fraction) was collected and frozen in an ethanol-dry ice bath. The pellet was re-suspended in 2 ml of 1:10 diluted B-PER reagent (PIERCE). The suspension was centrifuged and washed one more time with 1:10 diluted B-PER reagent. The pellet was re-suspended in 1 ml of Inclusion Body Solubilization Reagent. The suspension was vortexed for 1 min, shaken for 30 min, and sonicated to further break the cellular structures. Cell debris was removed from the suspension by centrifugation. The supernatant was frozen in an ethanol-dry ice bath (low solubility fraction).

Mass spectrometry sample preparation

The low solubility and high solubility protein fractions were subjected to buffer exchange, thiol reduction and alkylation, and tryptic digestion by the filter-aided sample preparation (FASP) procedure [23]. Peptides were desalted using a home-packed capillary reversed-phase column (500 μm internal diameter × 20 cm, POROS 10R2 C18 resin) using a Harvard syringe pump, and eluted directly onto a home-packed capillary strong cation exchange column (500 μm internal diameter × 45 cm, POROS SCX resin ), which was connected to the outlet of the reversed-phase column to minimize sample loss. SCX fractionation of peptides was conducted using a Dionex Ultimate NanoLC capillary HPLC system (Dionex, Sunnyvale, CA, USA), using a gradient from a 75%:25% mix of buffers A:B to 100% buffer B (buffer A, 7 mM KH2PO4, pH 2.65, 30% acetonitrile (ACN); buffer B, 7 mM KH2PO4, 350 mM KCl, pH 2.65, 30% ACN) over an 80-min period at flow rate of 10 μl/min, followed by column stripping and reconditioning for 10 min in buffer C (50 mM K2HPO4, 500 mM NaCl, pH 7.5) and water. SCX fractions were collected every 5 min and were pooled into 12 fractions of roughly equivalent peptide abundance according to the integration of their UV absorbance (λ = 214 nm) values during the course of separation.

Mass spectrometry data acquisition

Fractions were desalted using StageTip micro-scale reversed-phase chromatography [24], then subjected to reversed-phase nano-LC-MS and MS/MS performed on a nano-flow capillary high pressure HPLC system (Eksigent, Dublin, CA, USA) coupled to an LTQ-Orbitrap hybrid mass spectrometer (ThermoFisher Scientific, San Jose, CA, USA). Sample concentration and desalting were performed online using a trapping capillary column (200 μm × approximately 30 mm, packed with 5 μm, 100 Å Magic AQ C18 material; Michrom, Auburn, CA, USA) at a flow rate of 7 μl/min for 3.5 min, while separation was achieved using an analytical capillary column (75 μm × approximately 20 cm, packed with 3 μm, 100 Å Magic AQ C18 material; Michrom) terminating in a pulled sprayer tip, under a linear gradient of A and B buffers (buffer A, 3% ACN/0.1% formic acid; buffer B, 97% ACN/0.1% formic acid) over 180 min at a flow rate of approximately 0.5 μl/min. Electrospray ionization was carried out at 2.5 kV, with the LTQ heated capillary set to 200°C. Full-scan mass spectra were acquired in the Orbitrap in the positive-ion mode over the m/z range of 300 to 1,800 at a resolution of 60,000. MS/MS spectra were simultaneously acquired using the LTQ for the seven most abundant multiply charged species in the full-scan spectrum having signal intensities of > 1,000 NL (Thermo). Dynamic exclusion was set such that MS/MS was acquired only once for each species over a period of 120 s.

Peptide identification and quantification

We used a database of peptide sequences for E. coli K-12 from the UniprotKB, which was built by in silico tryptic digest to obtain two databases of labeled and unlabeled peptide masses and nitrogen counts. XICs and their associated fragmentation spectra were detected using methods we previously developed [13]. Monoisotopic XICs were paired in order of decreasing intensity. During pairing, the mass determined from the mass-to-charge ratio (m/z) and charge z obtained from 13C isotope spacing was used to search both databases. A positive or negative shift of 0.99703489341 × N/z was examined for candidate XICs for each returned nitrogen count N for the unlabeled and labeled database, respectively. The m/z of the candidate XIC was allowed to vary between a ± 3 ppm window. Also, the pairing allowed the position of a candidate XIC to vary between the start and end time of the current XIC to compensate for differences in co-elution time known to occur due to 15N-labeling. If only a single candidate XIC was found, the current XIC and the candidate XIC were paired, and a labeling status and nitrogen count were assigned to any associated fragmentation spectra. Based on labeling status, fragmentation spectra were searched against an unlabeled or labeled tryptic peptide database using the fragmentation model from the Open Mass Spectrometry Search Algorithm [25] and scored according to (M/L) Σ log2 (Im), where L is the number of peaks in the theoretical spectrum, M is the number of matched experimentally observed peaks, and Im is the intensity of the m-th peak that matched a theoretical spectrum peak. This modified score was used to leverage intensity information in spectra and assure near Gaussian distribution of the scores. Prior to scoring, peptides with matching precursor masses but differing nitrogen counts were filtered. FDR was estimated using a q-value approach and concatenated reverse decoy database [26, 27]. XICs that have fragmentation spectra without a corresponding XIC pair, or an ambiguous pair, can be assigned an identification and labeling status based on database search of a labeled and 15N-labeled database.

We used the implementation of XPRESS in Trans Proteomic Pipeline (v.4.4.1) [12]. The mass tolerance for XPRESS was set to 0.05 Da. All tryptic peptides were searched using a precursor mass window of ± 10 ppm at a stringent FDR of 1%. A single missed cleavage was allowed during peptide database construction.

We have implemented our method in an open source software system for LC-MS data analysis that provides an extremely fast and highly efficient implementation of this method [13, 28]. A tutorial and all the raw mass spectra analyzed in this manuscript are available for download at [29] and at Proteome Commons [30] with the following Tranche Hash: 4b3VyisnIV6ZXYHRRebqqLxSpgy45DrDVqJojES07+m8JrGaL8RshZx6y/XFZNYSU1iL3VJY7mwhi3XvxhESYXOpBaYAAAAAAAACuQ = =

coefficient of variation

false discovery rate

liquid chromatography coupled mass spectrometry

tandem mass spectrometry

parts per million

extracted ion chromatogram

strong cation exchange

stable isotope labeling by amino acids in cell culture.

NIH grant P50 GM071508 (ZK, AAC, DHP, SA); James S McDonnell Centennial Fellowship (LK); Howard Hughes Medical Institute (LK); NIH grant 5R01AI077562 (ST). We would also like to thank Sean R Hackett and Ileana Cristea for their insightful comments on this manuscript.

Authors’ Affiliations

Department of Computer Science, Princeton University
Lewis-Sigler Institute for Integrative Genomics, Princeton University
Department of Human Genetics, University of Chicago
Department of Molecular Biology, Princeton University
Illumina Inc.
Cold Spring Harbor Laboratory
Donnelly Centre for Cellular and Biomolecular Research, University of Toronto
Department of Ecology and Evolutionary Biology, Princeton University
Howard Hughes Medical Institute, Princeton University
Princeton Mass Spectrometry Center, Princeton University
Department of Biochemistry and Molecular Biophysics, Columbia University

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© Khan et al.; licensee BioMed Central Ltd. 2011

This article is published under license to BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


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Geographic area: Designated Place
Geographic code: 130095

Note: Standard geographic areas are highlighted on the map and associated data is displayed in the data table.

Table search results for: Upper Gagetown, CANADA
Selected characteristics Current selection Previous selection(s): maximum 4
Place name (Geographic code)
Type Local service district ( LSD ) ...
Boundary DPL ...
Geographic code 130095
2011 Census
Population and dwelling counts
Population 316 33,476,688
Total private dwellings 146 14,569,633
Private dwellings occupied by usual residents 134 13,320,614
Age and sex Footnote 1
Total - Age and sex 32033,476,685
0 to 14 years 455,607,345
15 to 64 years 22522,924,285
65 years and over 504,945,060
Male 15016,414,225
Female 16017,062,460
Census family structure
Total number of census families 1109,389,700
Number of married-couple families 756,293,950
Number of common-law couple families 251,567,905
Number of lone-parent families 151,527,840
Number of female lone-parent families 101,200,295
Number of male lone-parent families 5327,550
Private household type
Total number of private households 13513,320,615
Households containing a couple (married or common-law) with children aged 24 and under at home 403,524,915
Households containing a couple (married or common-law) without children aged 24 and under at home Footnote 2 603,935,540
Lone-parent family households 101,375,450
One-person households 253,673,305
Other household types Footnote 3 5811,405
Average number of persons in private households 2.32.5
Marital status
Total population 15 years and over by marital status 27027,869,340
Married or living with a common-law partner 19516,084,490
Married (and not separated) 15012,941,960
Living common-law 453,142,525
Not married and not living with a common-law partner 8011,784,855
Single (never legally married) 557,816,045
Separated, but still legally married 5698,240
Divorced 51,686,035
Widowed 151,584,530
Occupied private dwelling characteristics
Total private dwellings occupied by usual residents 13513,320,615
Single-detached houses 1307,329,150
Semi-detached houses 0646,245
Row houses 0791,595
Apartments or flats in a duplex 0704,480
Apartments in a building with fewer than five storeys 02,397,550
Apartments in a building with five or more storeys 01,234,770
Other dwellings Footnote 4 0216,820
Language characteristics
Total population excluding institutional residents 31533,121,175
Mother tongue
English 30018,858,975
French 57,054,970
Non-official languages 56,567,685
Aboriginal languages 0200,725
Non-Aboriginal languages 56,366,960
English and French 0144,685
English and non-official language 0396,330
French and non-official language 074,430
English, French and non-official language 024,095
Knowledge of official languages
English only 29022,564,665
French only 04,165,015
English and French 255,795,575
Neither English nor French 5595,920
Language spoken most often at home
English 31021,457,075
French 56,827,865
Non-official languages 53,673,865
Aboriginal languages 0121,810
Non-Aboriginal languages 53,552,055
English and French 0131,205
English and non-official language 0875,135
French and non-official language 0109,705
English, French and non-official language 046,330
First official language spoken
English only 30524,662,895
French only 57,507,885
English and French 0367,635
Neither English nor French 0582,755

Footnote 1

Return to footnote 1 referrer All counts in census tabulations are subjected to random rounding. More information.

Footnote 2

Return to footnote 2 referrer 'Without children' includes households containing a couple with all children in the home aged 25 and over.

Footnote 3

Return to footnote 3 referrer 'Other' refers to two or more people who share a private household, but who do not consitute a census family.

Footnote 4

Return to footnote 4 referrer Includes other single-attached houses, mobile homes and other movable dwellings such as houseboats and railroad cars.

How to cite:
Statistics Canada. 2012. GeoSearch. 2011 Census. Statistics Canada Catalogue no. 92-142-XWE. Ottawa, Ontario. Data updated October 24, 2012.
(accessed 2016-08-28)


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For Immediate Release
Office of the Vice President
October 11, 2004

Vice President Cheney's Remarks at a Victory 2004 Rally in Batavia, Ohio
Eastern Cincinnati Aviation Hangar
Batavia, Ohio

2:35 P.M. EDT

THE VICE PRESIDENT: Thank you all. (Applause.) Thank you very much for that welcome. It's great to be back in Ohio again. And it looks to me like Batavia is Bush-Cheney country. (Applause.)

And it's true that Lynne has known me since I was 14 years old, but she wouldn't go out with me until I was 17. (Laughter.) I tell people we got married because Dwight Eisenhower got elected President of the United States. True story. In 1952, I was a youngster living in Lincoln, Nebraska with my folks. Dad worked for the Soil Conservation Service. Eisenhower got elected, reorganized the government, Dad got transferred to Casper, Wyoming. And that's where I met Lynne, and we grew up together, went to high school together, and recently celebrated our 40th wedding anniversary. (Applause.) I explained to a group the other night that if it hadn't been for Eisenhower's great election victory, Lynne would have married somebody else. (Laughter.) And she said, right, and now he'd be Vice President of the United States. (Laughter.) No doubt in my mind. (Laughter.)

Well, like most of you, Lynne and I watched the debate on Friday and thought the President did a fantastic job. (Applause.) Now, with 22 days left in the campaign, the choices in this election are very clear. The stakes are high, both at home and abroad. And I believe on November 2nd, the American people are going to make George W. Bush President for four more years. (Applause.)

The President and I are delighted to be running in Ohio with a solid slate of Republican candidates, first among them, of course, is your own congressman, Rob Portman. (Applause.) I served in the House of Representatives for 10 years, and I got to be a pretty good judge of congressional horse flesh. But I've got to tell you, in all my years of service, Rob Portman is right at the top of my list of competent, capable, dedicated public servants. (Applause.)

And he also makes a pretty good Joe Lieberman and John Edwards. (Laughter.) I better explain that. Because -- Rob has been great because the last two campaigns, this one and also four years ago, he turned and devoted a lot of effort to being my debating partner in the warm-up for those televised vice presidential debates. And I got to tell you, he was tougher than either one of them. (Laughter and applause.) I don't want to fault John Edwards. After all, it's pretty tough to defend John Kerry's positions when you don't know what they are. (Laughter and applause.)

But also up for reelection this year here in Ohio is George Voinovich. And I know George isn't here today, but he's served your state extremely well in the United States Senate. And he's well on his way to another term in the United States Senate. (Applause.)

And I want to mention two other old friends of mine. Mike DeWine and Governor Bob Taft couldn't be here today, but I enjoy very much and appreciate and respect their service to the nation, as well.

I had a great time debating Senator Edwards last Tuesday. (Applause.) People tell me that Senator Edwards got picked because of his good looks, his charm, his great hair, his sex appeal. (Laughter.) I said, "How do you think I got the job?" (Laughter.) Why do they laugh when I say that? (Laughter.)

But in all seriousness, this is a very important election -? and it could not come at a more crucial time in our history. Today we face an enemy today every bit as intent on destroying us as the Axis powers were in World War II. This is not an enemy we can reason with or negotiate with or appease. This is, to put it simply, an enemy that we must destroy. And with George Bush as Commander-in-Chief, that is exactly what we are doing. (Applause.)

Under President Bush's leadership, we are confronting the terrorists with our military, so we do not have to fight them with armies of firefighters, police and medical personnel on the streets of our own cities.

Since the attacks of September 11th, President Bush has led a clear, steady, and consistent effort to protect the American people. We are going after the terrorists wherever they train and hide. And we are confronting regimes that sponsor terrorists or give them safe haven. And in the broader Middle East, we're aiding the rise of democracy, because free nations will not be the breeding grounds of terror.

We're making progress. We have ended the Taliban regime. And Saddam Hussein is in jail. (Applause.) We have broken up terror cells around the world, and captured or killed thousands of al Qaeda. We're training security forces in both Afghanistan and Iraq and we're rebuilding schools and hospitals to improve lives. And we're helping the people of Afghanistan and Iraq to build representative, democratically elected governments. Afghanistan, where almost half of the 10 million registered voters are women, held its first democratic election in history on Saturday. (Applause.) And Iraq will have democratically -- democratic elections next January. This is absolutely vital to completing the task of making certain that those nations don't ever again become breeding grounds for terror, or for the development and use of weapons of mass destruction.

President Bush does not deal in empty threats and half-way measures, and his determination has sent a clear message. Just five days after Saddam was captured, the government of Libya agreed to abandon its nuclear weapons program and turn the materials over to the United States. (Applause.) Today, the uranium, the centrifuges, and the designs for nuclear weapons that once were hidden in Libya are locked up and stored away, never again to be a danger to Americans.

The President is determined to prevail in the global war on terror, but even after 9/11, John Kerry has often seemed not quite to understand the threat. From 9/11 to this hour, our principal concern has had to be that the terrorists will strike again, and that this time they will try use even deadlier weapons. We know they seek chemical, biological and nuclear capability, which means we cannot wait until we are attacked to deal with them, as Senator Kerry suggested in his speech at the Democratic Convention. Nor can we think of our goal in this war in the way Senator Kerry described it yesterday in The New York Times. "We have to get back to the place," he said, where terrorism is "a nuisance," sort of like -? and these are his comparisons -- sort of like gambling and prostitution.

THE VICE PRESIDENT: This is naive and dangerous, as was Senator Kerry's reluctance earlier this year to call the war on terror an actual war. He preferred to think of it, he said, as primarily an intelligence and law enforcement operation. This is all part of a pre-9/11 mind set, and it is a view that we cannot go back to.

The 9/11 attack was the worst ever on American soil. We lost more people than at Pearl Harbor. Since then, we've seen attacks all over the world -- in Madrid, Casablanca, Mombassa, Riyadh, Istanbul, Jakarta, Bali, Baghdad, Beslan in Russia, and most recently in Egypt. This is a global conflict. If we fail to aggressively prosecute the war on terror, destroying terrorists where we find them and confronting governments that sponsor terror, the danger will only increase. The terrorists will escalate their attacks, both here at home and overseas, and the likelihood will increase that they will acquire weapons of mass destruction to use against us. Ultimately the cost of dealing with this threat will be far higher than confronting it now.

Something Senator Kerry said in the first presidential debate reveals a similar mind set, the same lack of understanding of the danger we face. He said that before America acts, we must pass a quote "global test."

THE VICE PRESIDENT: The President and I know, even as we work to build international alliances around the world, that our job is not to conduct international opinion polls. Our job is to protect the American people. And we will never -- (Applause.) We will never seek a permission slip to defend the United States of America. (Applause.)

When John Kerry suggests a global test, he goes right back to his beginnings in politics, when he said as he ran for Congress the first time, he would only deploy troops under the authority of the United Nations.

THE VICE PRESIDENT: During the 1980s, he opposed Ronald Reagan's major defense initiatives that brought victory in the Cold War. In 1991, when Saddam Hussein occupied Kuwait and stood poised to dominate the Persian Gulf, Senator Kerry voted against Operation Desert Storm. You occasionally hear some bold talk from him, but it cannot disguise a 30-year record of coming down on the wrong side of virtually every major defense issue. (Applause.)

Perhaps Senator Kerry's most notorious vote is the one against funding for our troops. After voting for the use of force in Iraq, he voted against the $87 billion needed by our men and women in uniform for ammunition, spare parts, and fuel. The reason for his shift was simple: Howard Dean, the anti-war Democrat in the Democratic primaries, was surging ahead. Now, I have to ask: if Senator Kerry can't stand up to the pressures posed by Howard Dean, how can we expect him to stand up to the al Qaeda? (Applause.)

The other night in the debate, Senator Kerry said, and I quote, "I've never changed my mind about Iraq." (Laughter.) "I do believe Saddam Hussein was a threat." End quote. And then within minutes he changed his mind, saying that Iraq, and I quote "wasn't a threat." The Senator, who likes to say he never wavers, wavered within the course of a single debate, and that matters, because our troops, our allies, and our enemies must know where America stands. (Applause.)

The President of the United States must be clear and consistent. The Iraqi people need to know that America will always keep its promises. The terrorists need to know that we will not cut and run. And our men and women in uniform need to know that we will honor their service and sacrifice by completing the mission. (Applause.)

President Bush knows that our dedicated servicemen and women represent the very best of the United States of America. And I want to thank them, and their families, and all the veterans here with us today for what they've done for all of us. (Applause.) One of the most important commitments that the President made during the 2000 campaign was that our armed forces would be given the resources they need and the respect they deserve, and he has kept his word to the U.S. military. (Applause.)

In his years in Washington, John Kerry has been one of a hundred votes in the United States Senate -? and very fortunately on matters of national security, his views rarely prevailed. A senator can be wrong; a senator can be confused; a senator can be indecisive for 20 years without consequence to the nation. But a President always casts the deciding vote. And in this time of challenge, America needs -? and America has -? a President we can count on to get it right. (Applause.)

Senator Kerry's back-and-forth reflects a habit of indecision, and sends a message of confusion. And it's all part of a pattern. He has, in the last several years, been for the No Child Left Behind Act -? and against it. He has spoken in favor of the North American Free Trade Agreement -? and against it. He is for the Patriot Act -? and against it. Senator Kerry says he sees two Americas. It makes the whole thing mutual ?- America sees two John Kerrys. (Applause.)

Our country requires strong and consistent leadership for our actions overseas, as well as for our policies here at home. When President Bush and I took the oath of office on the inaugural platform on the west front of the Capitol, our economy was sliding into recession. Then, on 9/11, terrorists struck and shook our economy once again. The President responded by delivering tax cuts four times in four years.

Every American who pays federal income taxes benefited from the Bush tax cuts -? and so has our economy. We've created jobs for 13 consecutive months, a total of over 1.9 million new jobs during that period. (Applause.) Mortgage rates, interest rates and inflation are all low. Consumers are confident. Businesses are investing, families taking home more of what they earn. We're seeing record exports for farm products. Farm income is up. Our farm economy is strong. That's good for the entire nation.

We know there are still challenges, especially in our manufacturing communities. The President and I will not be satisfied until every American who wants to work can find a job. (Applause.) We plan to double the number of workers trained through the federal government's job training program. We've proposed a quarter of a billion dollars to help more workers train at our nation's fine community colleges. We'll improve math and science education in our public high schools so that every high school graduate gets the quality education they deserve and the foundation they need to fill the jobs of the 21st century.

Our accomplishments these past four years have made America safer, stronger, and better. They also demonstrate something about the character of our President. He didn't go to the White House to mark time and spend his energy on small goals. He went to take on the big issues, and to make serious reforms. He has led with confidence, with clear vision, and unwavering purpose. He's made hard choices, and he's kept his word. And that's exactly how he will continue to lead the country for the next four years. (Applause.)

In our second term, we'll keep moving forward with a pro-growth, pro-jobs agenda. We'll work to make the Bush tax cuts permanent. (Applause.) And to help families and small businesses, we'll lead a bipartisan effort to reform and simplify the federal tax code. (Applause.)

We will work to end lawsuit abuse. (Applause.) We know that it's a lot easier for America's businesses to hire new workers if they don't have to keep hiring lawyers. (Applause.)

We will work for medical liability reform because we know the cost of malpractice insurance is creating a crisis, not only in Ohio, but all across the nation. America's doctors should be able to spend their time healing patients, not fighting off frivolous lawsuits. (Applause.)

Our opponents have a very different vision for our country. In the Senate, John Kerry voted to increase taxes 98 times. He opposed the President's middle class tax relief, and he voted to squeeze another $2,000 per year from the average middle class family. He is opposed to reform of our legal system, and he is against medical liability reform. Now Senator Kerry is proposing massive increases in federal spending. His big idea for the economy: raise our taxes.

THE VICE PRESIDENT: That's all right. (Laughter.) President Bush and I will also continue to defend our society's fundamental rights and values. We stand for a culture of life and reject the brutal practice of partial birth abortion. (Applause.) We stand strongly for the Second Amendment and will defend the individual right of every American to bear arms. (Applause.) We believe that our nation is "one nation under God." (Applause.) And we believe Americans ought to be able to say so when we pledge allegiance to our flag. (Applause.)

There shouldn't be any question about this -? and there wouldn't be if we had more reasonable judges on the federal bench. The Democrats in the Senate have been doing everything they can -? including using the filibuster ?- to keep the President's sensible, mainstream nominees off the bench.

THE VICE PRESIDENT: They are hoping to wait the President out. But I've got news for them. That's not going to happen. We're going to win this election. (Applause.)

AUDIENCE: Four more years! Four more years! Four more years!

THE VICE PRESIDENT: And a good way to deal with the problem of the Democratic filibuster in the Senate is to reelected good Republicans like George Voinovich. (Applause.)

My friends, the differences between the President and his opponent are as sharp as they can possibly be, and the consequences for the country are enormous. On vital matters of national security, Senator Kennedy -- excuse me. (Laughter and applause.) I keep forgetting that Senator Kerry is the more liberal of the two. (Laughter.)

The Senator offers a record of weakness and a strategy of retreat on defense issues. President Bush offers a record of steady purpose and resolute action, and a strategy for victory. Senator Kerry is a tax-and-spend liberal; President Bush, a compassionate conservative. Senator Kerry wants to empower government; President Bush will use government to empower people. (Applause.) John Kerry seems to think that all the wisdom is found in Washington, D.C.; George Bush trusts the wisdom of the American people. (Applause.)

On issue after issue, President Bush has a clear vision for the future of our nation. America has come to know him, and I have come to admire him very much. I watch him at work every day. He's a person of loyalty and kindness, a man who speaks plainly and means what he says. I have seen him face some of the hardest decisions that can come to the occupant of the Oval Office -? and make those decisions with the wisdom and the humility Americans expect in their President.

Under President Bush's leadership, we will use America's great power to serve great purposes, to protect our homeland by turning back and defeating the forces of terror, and to spread hope and freedom around the world. Here at home, we'll continue building a prosperity that reaches every corner of the land so that every child in America has a chance to learn, to succeed, and to rise in the world. (Applause.)

The President and I are honored by your commitment to the cause we all share. President Bush and I will wage this effort with complete confidence in the American people. The signs are good -? here in Ohio, and even in Massachusetts. (Applause.) According to news accounts, people leaving the Democratic National Convention in July asked a Boston policeman for directions. He replied, "Leave here -? and go vote Republican." (Laughter and applause.)

President Bush and I are honored to have the support of that police officer, and of Democrats, Republicans, and independents from every calling in American life. We're grateful to our many friends across the great state of Ohio. I want to thank you for this tremendous welcome today. We're proud to have you on the team. And together, on November 2nd, we'll see our cause forward to victory.

Thank you very much. (Applause.)

END 2:59 P.M. EDT

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Attached files

file filename
EX-99.1 - GRIFFON CORPc71580_ex99-1.htm

Washington, D.C. 20549

Pursuant to Section 13 or 15(d) of the

Securities Exchange Act of 1934

Date of Report (Date of earliest event reported): November 13, 2012

(Exact Name of Registrant as Specified in Charter)

Delaware 1-06620 11-1893410
(State or Other Jurisdiction (Commission (I.R.S. Employer
of Incorporation) File Number) Identification Number)

712 Fifth Avenue, 18th Floor
New York, New York   10019
(Address of Principal Executive Offices)   (Zip Code)

(212) 957-5000

(Registrant’s telephone number, including area code)

Check the appropriate box below if the Form 8-K filing is intended to simultaneously satisfy the filing obligation of the registrant under any of the following provisions (see General Instruction A.2. below):

[ ] Written communications pursuant to Rule 425 under the Securities Act (17 CFR 230.425)

[ ] Soliciting material pursuant to Rule 14a-12 under the Exchange Act (17 CFR 240.14a-12)

[ ] Pre-commencement communications pursuant to Rule 14d-2(b) under the Exchange Act (17 CFR 240.14d-2(b))

[ ] Pre-commencement communications pursuant to Rule 13e-4(c) under the Exchange Act (17 CFR 240.13e-4(c))

Item 2.02.      Results of Operations and Financial Condition.

On November 13, Griffon Corporation (the “Registrant”) issued a press release announcing the Registrant’s financial results for the fourth fiscal quarter and full year ended September 30, 2012. A copy of the Registrant’s press release is attached hereto as Exhibit 99.1.

Item 9.01.      Financial Statements and Exhibits.

(d)        Exhibits.

99.1      Press Release, dated November 13, 2012

The information filed as an exhibit to this Form 8-K is being furnished in accordance with Item 2.02 and shall not be deemed to be “filed” for the purposes of Section 18 of the Securities Exchange Act of 1934, as amended (the “Exchange Act”), or otherwise subject to the liabilities of such section, nor shall such information be deemed incorporated by reference in any filing under the Securities Act of 1933, as amended, or the Exchange Act, except as shall be expressly set forth by specific reference in such a filing.

Pursuant to the requirements of the Securities Exchange Act of 1934, the Registrant has duly caused this report to be signed on its behalf by the undersigned hereunto duly authorized.

  By:  /s/ Douglas J. Wetmore
        Douglas J. Wetmore
    Executive Vice President and
    Chief Financial Officer

Date: November 13, 2012

Exhibit Index

99.1Press release, dated November 13, 2012


text:
ggFTW (
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Griever 01-14-2011 11:36 AM

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