Saturday, June 2, 2012

Enhanced Identification of Peptides Lacking Basic Residues

The new issue of Proteomics has 2 articles in it that I find really interesting.  The first is by Martin Biniossek and Oliver Schilling of the BIOSS Center.  This paper begins to address one of the gaping holes in shotgun proteomics, the requirement that peptides are multiply-charged.  I know I wrote about this once before, but it may be in an article that I haven't transferred from the old blog.
Anyway, in most cases, your peptide must have more than 1 charge on it ore you won't get a good fragmentation pattern.  The MS/MS spectra will show only the fragments that maintained the charge.  For example, if the terminal amino acid on one side of your peptide is charged, you will only see fragments that contain that amino acid.  Since half of the potential fragments are now invisible, you probably won't get enough information from the MS/MS spectra to accurately sequence that ion.  There are other reasons for investigating doubly charged ions that I go into in that missing post.  When I find it, I'll insert it here.
This paper attempts to fill that hole by studying the MS/MS spectra of singly charged ions.  Their motivation is the fact that the use of some alternative proteolytic enzymes result in fewer multiply charged peptides than you get with a tryptic digest.  The real emphasis of this project was to determine if the analysis of singly charged peptides would increase the coverage of peptides lacking a basic residue.

For this study they used two enzymes, GluC and ChymoTrypsin, to digest the proteome of E.coli strain MG1655.  Since these enzymes do not cut at basic residues, the peptides are less likely to multiply-charge.  The proteome was also digested with trypsin, for comparison's sake.

The MS/MS analysis was performed with a QStar Pulsar coupled to an Ultimate 3000 system.
The experiment was done in three ways:
1) Ions were only selected for MS/MS if they were singly charged
2) Ions were only selected for MS/MS if they were multiply charged
3) Ions were fragmented regardless of charge state

As expected, experiment 1 resulted in low peptide coverage, with the chymotrypsin digest producing the most identified peptides, 64.  However, >95% of the peptides identified from the chymotrypsin and GluC digests were found to be the peptides of interest, the ones lacking basic residues.
Experiment 2, the classical approach resulted in the largest number of ID'ed proteins, with trypsin the clear leader at 1108 peptides.
Experiment 3 resulted in fewer peptide IDs, with the highest number again coming from trypsin, but only 989 were ID'ed.  This is surprising at first, until you think about it.  I am sure that more ions were selected for fragmentation in the third experiment than in the second.  Unfortunately, only a small percentage of the singly charged ions could be identified with high confidence.  I have tried similar experiments twice in the past with virtually the same results.  The number of fragment ions goes through the roof, but the number of peptides ID'ed decreases.

In summary:  This paper shows the promise of targeting singly charged peptides for increasing the coverage of peptides lacking basic residues.  It is a quick read and an elegant approach to this problem, and ultimately a nice first step toward addressing a key weakness in our field.

No comments:

Post a Comment