Proteome discoverer platform

The
raw files acquired by the MS system were processed using the Proteome
Discoverer platform (version 1.4, Thermo Scientific). An integrated
workflow using the algorithms Sequest HT and Mascot (version 2.4,
Matrix Science) was employed. Either a human UniProtKB database (Release
2013_6; 88 295 human sequences) or a database consisting of
the aforementioned human proteins and all protein sequences derived
from 21 microbial genomes (Supporting Information Table S-1) were used. The latter database was used to identify human
and microbial proteins present in UP samples. MS search parameters
similar to published previously²⁷ are described
in detail in Supporting Information. For
protein quantification of the data sets, the MaxQuant software suite
(version 1.4.2) was used.^{32 (link)} Most of the
default settings provided in this software suite were accepted, and
data were processed using both the label-free quantitation (LFQ) and
the intensity-based absolute quantitation (iBAQ) tools. The LFQ algorithms
provide relative quantification of the integrated MS¹ peak
areas from the high resolution MS data. The iBAQ algorithms sum the
integrated peak intensities of the peptide ions for a given protein
divided by the number of theoretically observable peptides, which
are calculated by in silico digestion of protein sequences including
all fully tryptic peptides with a length of 6–30 amino acids.^{33 (link)}