Bingwen Lu

USHUPO 2009 just finished days ago here in San Diego. I attended this conference and here are what I thought some gems from the conference.

• Albert Heck from The Netherlands presented the use of a new endoprotease LysN combining with ETD mass spectrometer for de novo peptide sequencing. They got nice ladders of C ions that could be read easily to derive sequences.
• Christine Wu from University of Colorado presented a talk on doing LC at elevated temperature (60 degree C) for better separation and identification of membrane peptides and proteins. This is not totally new. Last time in 2007 they showed identification of ~100 proteins but this time they scaled up to ~1,100 proteins.
• Brian Halligan from Medical College of Wisconsin presented an implementation of Amazon Web Services (AWS) for tandem mass spectrometry data analysis using XTandem and OMSSA. See here for more details. The cost (charged by Amazon.com) for analyzing each data set is $0.84 (will depend on size of data), according to what they said. That could be quite helpful for some laboratories that do not have large computational power. I think I will try it out sometime.

Just a side note, I did not go to all the sessions, so I might miss some excellent talks. Nevertheless, I was impressed and entertained by these three talks.

Following is a joke I translated from Chinese. It is related to a remark made today by Horace Engdahl, a figure of authority at the Swedish Academy, regarding the 2008 Nobel Prize for Literature. He commented that the Americans are too isolated from the world in term of literature.

Actually, different parts of the world are so quite different. Many times I am feeling very lucky and blessed, comparing with people from other corners of the world.

OK, here is the joke:

Reporter: What is your personal opinion on international food deficiency?

American: What is international?
Swiss: What is deficiency?
Ethiopian: What is food?
Cuban: What is personal?
North Korean: What is opinion?

Reference

http://www.cafebabel.com/eng/article/26676/nobel-literature-prize-horace-engdahl.html

One powerful thing is called attitude. After reading the following paragraph by Claude Hopkins, I think I can not agree more than what he said. Work is a play, isn’t it?

“If a thing is useful they call it work, if useless they call it play. One is as hard as the other. One can be just as much a game as the other… All the difference I see lies in attitude of mind… I came to love work as other men love golf. I love it still. Many a time I beg off from a bridge game, a dinner, or a dance to spend the evening in my office. I steal away from weekend parties at my country home to enjoy a few hours at my typewriter.” –Claude C. Hopkins

This post is written mostly for people who are new to the proteomics field.

Spectral counts are the number of tandem mass spectra (MS/MS) that are matched to the peptides for a protein. It is sometimes used as an indicator of protein abundance. Spectral counting is one widely used label-free protein quantitation method by mass spectrometry approach. It was first proposed by Liu et al (2004).

Example: For the following protein:
>gi|30794280|ref|NP_851335.1| albumin [Bos taurus]
MKWVTFISLLLLFSSAYSRGVFRRDTHKSEIAHRFK
DLGEEHFKGLVLIAFSQYLQQCPFDEHVKLVNELTE
FAKTCVADESHAGCEKSLHTLFGDELCKVASLRET
YGDMADCCEKQEPERNECFLSHKDDSPDLPKLKPD
PNTLCDEFKADEKKFWGKYLYEIARRHPYFYAPELL
YYANKYNGVFQECCQAEDKGACLLPKIETMREKVL
TSSARQRLRCASIQKFGERALKAWSVARLSQKFPK
AEFVEVTKLVTDLTKVHKECCHGDLLECADDRADLA
KYICDNQDTISSKLKECCDKPLLEKSHCIAEVEKDAI
PENLPPLTADFAEDKDVCKNYQEAKDAFLGSFLYEY
SRRHPEYAVSVLLRLAKEYEATLEECCAKDDPHACY
STVFDKLKHLVDEPQNLIKQNCDQFEKLGEYGFQNA
LIVRYTRKVPQVSTPTLVEVSRSLGKVGTRCCTKPE
SERMPCTEDYLSLILNRLCVLHEKTPVSEKVTKCCT
ESLVNRRPCFSALTPDETYVPKAFDEKLFTFHADIC
TLPDTEKQIKKQTALVELLKHKPKATEEQLKTVMEN
FVAFVDKCCAADDKEACFAVEGPKLVVSTQTALA

A tandem mass spectrometry experiment identified the peptides “GVFRRDTHKSEIAHRFK” and “EYEATLEECCAK”. The peptide “GVFRRDTHKSEIAHRFK” was matched by 3 separate tandem mass spectra, while the peptide “EYEATLEECCAK” was matched by 2 separate tandem mass spectra. No other peptide from this protein was matched by tandem mass spectra. The overall spectral counts for this protein is 2+3 = 5.

Reference

Liu H, Sadygov RG, Yates JR 3rd. A model for random sampling and estimation of relative protein abundance in shotgun proteomics. Anal Chem. 2004 Jul 15;76(14):4193-201.

Spectral counting can be used for protein quantification in two scenarios.

In the first scenario, protein abundances are compared across experiments for the same proteins. For example, the abundance of Protein1 in experiment S1 is compared to that of experiment S2. Spectral counts normalization is relatively simple in this scenario – we just need to make sure that after normalization, the sum of total spectral counts t1 for all the identified proteins in experiment S1 equals the total spectral counts t2 for all the identified proteins in experiment S2. One straightforward normalization formula is, for each protein, the normalized spectral count f1 for the protein in experiment S1 and f2 for the protein in experiment S2 are,

where n1 is the un-normalized spectral count for the protein in experiment S1, n2 is the un-normalized spectral count for the same protein in experiment S2.

In the second scenario, protein abundances are compared within a single experiment. For example, the abundance of Protein1 is compared to the abundance of Protein2. In this scenario, it was suggested that protein length should also be taken into consideration when doing spectral counting normalization (Zybailov et al., 2006). The reason is that, for proteins of the same abundance, longer protein might have more number of spectral counts. In such a scenario, the following formula can be used for normalization:

where L is the length of the protein. Using such formulas, the sum of all f1 and the sum of all f2 will each equal to 1. Note that because the sum of all f1 equals the sum of all f2 (=1), this normalization can also be used to compare spectral counts across experiments.

The normalized spectral counts will then be used by statistical methods to see if the abundance is different for the interested protein(s). To avoid taking logarithm on zero’s when performing statistical tests, a special measure need to be taken. This special measure is to add a “pseudo count” to the spectral count for each protein. After considering pseudo count, the above formulas are now as following:

For comparing protein abundances only across experiments:

For comparing protein abundances within a single experiment or across experiments:

Reference

Zybailov B, Mosley AL, Sardiu ME, Coleman MK, Florens L, Washburn MP. (2006) Statistical analysis of membrane proteome expression changes in Saccharomyces cerevisiae. J Proteome Res. 5: 2339-2347.

Also see Tutorial on protein quantification using spectral counting G test

When WordPress is installed at the root folder, and the permalink is set up using only “postname”, it might be impossible to access subfolders under the root folder, as stated by Using WordPress Permalink.

I figured out a way to deal with such a situation. My solution is to create a subdomain with the same name as that of the subfolder. The subfolder now can be accessed through the subdomain.

For example, it is impossible for me to access my subfolder http://www.lubw.com/files/. However, it is fine for me to access my subdomain http://files.lubw.com/.

Two random quotes on how to be successful.

“When you confront a problem, you begin to solve it.” — Rudy Giuliani (Former Mayor of NY City)

“I’ve found that luck is quite predictable. If you want more luck, take more chances, be more active, show up more often.” — Brian Tracy

In this world, there are always problems to be solved. This website, InnoCentive, provides an opportunity for people to expose their problems to a large pool of people of different expertise all over the world.

And if you are someone who likes challenging problems, you can sign up as a problem solver to try to solve other people’s problem. It is even better that if you really solve other people’s problems, you will win cash awards. The awards are ranging from US$5,000 to US$1,000,000.

References

http://www.innocentive.com/

NY Times: If you have a problem, ask everyone

The R21 grant is a type of United States National Institute of Health Grant. R21 is intended to encourage exploratory/developmental research projects by providing support for the early and conceptual stages of development.

Some Characteristics of R21 Application

• Project period can be of up to two years
• The combined budget for direct costs for the two year project period may not exceed $275,000.
• R21 grant support is for new projects only; competing renewal applications will not be accepted.
• Two resubmissions of a previously reviewed exploratory/developmental grant application may be submitted as defined in NIH Policy at http://grants.nih.gov/grants/policy/amendedapps.htm.
• Introduction (required for a resubmission application) is limited to one page.
• Items 2-5 of the Research Plan (Specific Aims, Background and Significance, Preliminary Studies, and Research Design and Methods) may not exceed a total of 15 pages.
• No preliminary data is required but may be included if available.
• A Progress Report is not needed.
• Use the instructions for the appendix detailed in the SF424 (R&R) except that no more than 5 manuscripts, previously accepted for publication, may be included.

Reference
NIH Exploratory/Developmental Research Grant Award - R21 Grant

To compare protein expression between two experiments using spectral counting G-test, we use the chi-square table with one degree of freedom. The significance is assessed according to the upper critical values of chi-square distribution. Following we give the chi-square tables with different kinds of degrees of freedom at various critical values. For more information on “chi-square tables”, please see http://www.itl.nist.gov/div898/handbook/eda/section3/eda3674.htm

Upper critical values of chi-square distribution with n degrees of freedom.

Probability of exceeding the critical value:

Lower critical values of chi-square distribution with n degrees of freedom

Probability of exceeding the critical value

Reference

http://www.itl.nist.gov/div898/handbook/eda/section3/eda3674.htm