The remaining proteins were digested in trypsin overnight, using a simple FASP-like method in an Amicon filter with a 10 kD MW cutoff.
The peptides were desalted by ziptip and 1 ug of peptides were loaded on a 30 cm MAGIC column from Proteomics Plus, running at 250 nL/min. A standard top 10 method (CID) was used on an Orbitrap Velos with a dynamic exclusion after 2 occurrences. Only ions with >1 charge were used, with a m/z of 350-2000. The data was processed in Proteome Discoverer 1.3 using Mascot and Sequest with Percolator rescoring
1348 unique fragmentation events resulted in 956 high confidence peptide IDs (71% identification rate)
I have been curious for a long time about the distribution of useful ions. The real question is, I guess, am I wasting time? Are there any peptides in the low and high mass ranges, and if not, why am I scanning all the way to 2,000 on every MS/MS?
The average positively ID'ed peptide m/z was: 890.45, with a median of 892.98
The average fragment m/z was: 856.61, with a median of 836.96
What does the actual distribution look like?
Wow. Keep in mind that the I started at a m/z of 350, but its pretty obvious that we don't get a lot of ID'ed peptides from the lower m/z range. Nor do we see anything in the high mass range, with only 1 peptide ID'ed with an m/z >1600.
Does the distribution of fragmented ions look the same?
How do the two overlap? If the totals from each chart are adjusted to 100%, the distribution looks like this:
These results beg further analysis, and the first questions I have when looking at this are: 1) is this reproducible, or simply a single occurrence in mouse serum and 2) is this a consequence of using the FT for MS and IT for MS/MS? I'm going to look at 2 experiments where we performed FT-IT and FT-FT (HCD) analysis of the same samples.
The real take away message here is that we may be wasting precious scanning time in the +1600 m/z range in an FT-IT experiment that is reaping no benefits to our research