Correction of Errors inSpectrum Extraction Enhances Phosphopeptide Identification

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Omics
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Correction of Errors inSpectrum Extraction Enhances Phosphopeptide Identification

Postby Omics » Wed Mar 12, 2014 7:47 pm

Spectrum extraction is a critical processing step before database searches in proteomics, and errors in spectrum extraction result in the failure of peptide identification or false positive identifications. Evaluation of 7 commonly used tools indicates that even the best one makes many mistakes in spectrum extraction. Correction of errors in spectrum extraction improves both the sensitivity and confidence of phosphopeptide identification and the identification of peptides with other PTMs. The identification of unmodified peptides for protein identification&quantification benefits from the error correction as well.

http://pubs.acs.org/doi/abs/10.1021/pr4004486?journalCode=jprobs

Kelstrup
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Postby Kelstrup » Mon Mar 17, 2014 2:22 am

I am a bit surprised that fulll MS scans were acquired in centroid mode when Thermo's centroiding software is somewhat lacking? Anyone know why one would do that, especially for a purpose such as this? Disc space is cheap nowadays compared to an instrument I think?

Second question: These tools may have settings for recognizing peak shapes (I know MaxQuant and Raw2MSM do) - could this somehow be optimized for the given setting? Sometimes space-charging or other non-linear effects make the default settings less optimal.

Otherwise, I liked the study goal. Not enough people are looking at the data any more :)

Omics
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Postby Omics » Mon Mar 17, 2014 8:57 am

Thanks Kelstrup for your very careful reading.

Answer to 1st question: For LTQFT, we have recorded the full MS scan in centroid mode to save disk space since 2006 when the disk was still expensive. I understand your concerns about the effect of data recording mode. Actually, we also applied the same strategy to high-resolution proteomics and phosphoproteomics data recorded in profile mode (both MS1 and MS2) from latest Q Exactive, the conclusion remains the same, i.e. correction of spectrum extraction errors results in a siginifcant increase of peptide identification. We did not include it in this paper since it did not affect the conclusion, but definitely increase the length of the paper.


Answer to your 2nd question: It is true that MaxQuant and Raw2MSM are designed specially for high-resolution data recorded in profile mode. However, according to our test, they performs equally well on data recorded in centroid mode. For example, the accuracy of the monoisotopic peak selection is over 90%, better than other tested software. As you know, only a few parameters can be modified in these two software. We evaluated it and selected the best if it is not the default.

Kelstrup wrote:I am a bit surprised that fulll MS scans were acquired in centroid mode when Thermo's centroiding software is somewhat lacking? Anyone know why one would do that, especially for a purpose such as this? Disc space is cheap nowadays compared to an instrument I think?

Second question: These tools may have settings for recognizing peak shapes (I know MaxQuant and Raw2MSM do) - could this somehow be optimized for the given setting? Sometimes space-charging or other non-linear effects make the default settings less optimal.

Otherwise, I liked the study goal. Not enough people are looking at the data any more :)

Kelstrup
Angiotensin Member
Angiotensin Member
Posts: 35
Joined: Fri Oct 05, 2012 5:19 am

Postby Kelstrup » Tue Mar 18, 2014 12:47 am

Thanks for your answers. Really interesting :)

Omics
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Posts: 14
Joined: Sat Oct 06, 2012 10:34 am

Postby Omics » Thu May 15, 2014 7:03 pm

Good, thanks.


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