Computational and Mass-Spectrometry-Based Workflow for the Discovery and Validation of Missing Human Proteins: Application to Chromosomes 2 and 14

In
the framework of the C-HPP, our Franco-Swiss consortium has
adopted chromosomes 2 and 14, coding for a total of 382 missing proteins
(proteins for which evidence is lacking at protein level). Over the
last 4 years, the French proteomics infrastructure has collected high-quality
data sets from 40 human samples, including a series of rarely studied
cell lines, tissue types, and sample preparations. Here we described
a step-by-step strategy based on the use of bioinformatics screening
and subsequent mass spectrometry (MS)-based validation to identify
what were up to now missing proteins in these data sets. Screening
database search results (85 326 dat files) identified 58 of
the missing proteins (36 on chromosome 2 and 22 on chromosome 14)
by 83 unique peptides following the latest release of neXtProt (2014-09-19).
PSMs corresponding to these peptides were thoroughly examined by applying
two different MS-based criteria: peptide-level false discovery rate
calculation and expert PSM quality assessment. Synthetic peptides
were then produced and used to generate reference MS/MS spectra. A
spectral similarity score was then calculated for each pair of reference-endogenous
spectra and used as a third criterion for missing protein validation.
Finally, LC–SRM assays were developed to target proteotypic
peptides from four of the missing proteins detected in tissue/cell
samples, which were still available and for which sample preparation
could be reproduced. These LC–SRM assays unambiguously detected
the endogenous unique peptide for three of the proteins. For two of
these, identification was confirmed by additional proteotypic peptides.
We concluded that of the initial set of 58 proteins detected by the
bioinformatics screen, the consecutive MS-based validation criteria
led to propose the identification of 13 of these proteins (8 on chromosome
2 and 5 on chromosome 14) that passed at least two of the three MS-based
criteria. Thus, a rigorous step-by-step approach combining bioinformatics
screening and MS-based validation assays is particularly suitable to obtain protein-level evidence for proteins previously considered as missing. All MS/MS data have been deposited in ProteomeXchange under identifier PXD002131.