It should be emphasized that for this study we did not verify assignments by spiking natural samples with authentic commercial or synthetic standards, a common practice for final verification of compound identity by NMR.
Therefore, the first glycopeptide candidate, which is also the manually verified correct assignment, is assigned to the ETD data by GPE, even when four other incorrect candidates and 100 decoys are scored in parallel.
For cellulose-degrading species annotated in IMG, we verified these assignments based on these publications.
It is difficult to estimate the full dimension of this problem; nevertheless, a systematic rescue of those annotations lost in the literature is very much needed, given the low number of experimentally verified functional assignments in the current databases [ 78].
MS/MS of the doubly charged peptide cation and analysis of the internal fragments further verified its assignment, confirming the existence of the C22 C28 disulfide bond in L2 (Fig. 4B).
We also verified sex assignment of the samples by examining expression levels of genes on the Y chromosome.
Carefully verified manual assignment of E. coli gene products to functional categories was obtained from Biomax Informatics AG, Martinsried, Germany.
Manual inspection of MS/MS data was performed for proteins having only two peptide matches to verify sequence assignments.
It is noteworthy that, although CID data are utilized to verify glycopeptide assignments, in our method, we did not implement CID fragmentation rules in the scoring function, and only ETD data should be submitted to GPE for appropriate FDR analysis.
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