3.8 LC–MS/
MS Measurement
Peptides were loaded onto a Dionex RS3500 coupled to a Q
Exactive mass spectrometer. Peptides were loaded onto a 75μm
(ID), 15 cm column packed in-house with reversed-phase Phenomenex core-shell 1.7μm resin. Peptides were then eluted using a
50-min linear gradient of solvent B (100% ACN in 0.1% formic
acid) from 2% to 6% for 2 min and 6% to 23% for 48 min using curve
8 at 400 nL/min followed by a washout at 80% for 10 min. Full
scans were recorded between 300 and 1750 Thompson at a resolution of 70,000 with an AGC target of 3 Â 10
6 . For proteome
measurements, the 10 most intense ions from each full scan were
selected for fragmentation (MS/MS) by higher-energy collisional
dissociation (HCD) using an NCE of 28 and an AGC target of
1 Â 10
5 in 80 ms at a resolution of 17,500.
The raw data files were processed with the MaxQuant software
(version 1.6.1.0) using the Andromeda search engine to search
MS/MS data against UniProt Mycobacterium smegmatis (strain:
mc
2 155) (6602 entries) database with common contaminants.
Carbamidomethylation of cysteine (C) was set as fixed modification
and oxidation of methionine and NQ acetylation were defined as
variable modifications. Trypsin/P was selected as a protease with
2 missed cleavages allowed and at least two peptides were required
for LFQ quantitation. Precursor mass tolerance was 4.5 ppm and
MS/MS mass tolerance was 20 ppm for HCD fragmentation data
analysis. The false discovery rate (FDR) for peptides and proteins
was set to 1%. For all other parameters, the default settings were
used. Downstream statistical analysis was performed with Perseus
(1.6.10.45).
3.9 Protein
Identification and Data
Handling
Following MaxQuant-based protein identification and quantification, statistical analysis using Perseus software [7, 8] version
1.6.10.45. Protein identifications were filtered to only consider
proteins that had been identified with at least 2 unique peptides.
Reverse contaminants were also filtered out together with potential
contaminants. Single colonies were compared using analysis of
variance test (ANOVA) with p ¼ 0.05, FDR ¼ 0.05.
4 Results
Following MaxQuant-based spectral matching, 52,520 MS/MS
spectra were identified across all replicates. Cumulatively, 7805
unique peptides and 1387 unique proteins were identified at a
0.9% and 0.2% false discovery rate, respectively (Table 1). Following filtering, data distribution was normal (Fig. 1) and reproducibility was high (>94.7%) between replicates (Fig. 2), indicating
that the method detailed herein is highly reproducible. All raw and
spectral search output data were deposited to ProteomeXchange
(6) and are freely available under the identifier PXD018168.
Mass Spectrometry-Based Analysis of Mycobacterial Single-Colony Proteome
185
MS Measurement
Peptides were loaded onto a Dionex RS3500 coupled to a Q
Exactive mass spectrometer. Peptides were loaded onto a 75μm
(ID), 15 cm column packed in-house with reversed-phase Phenomenex core-shell 1.7μm resin. Peptides were then eluted using a
50-min linear gradient of solvent B (100% ACN in 0.1% formic
acid) from 2% to 6% for 2 min and 6% to 23% for 48 min using curve
8 at 400 nL/min followed by a washout at 80% for 10 min. Full
scans were recorded between 300 and 1750 Thompson at a resolution of 70,000 with an AGC target of 3 Â 10
6 . For proteome
measurements, the 10 most intense ions from each full scan were
selected for fragmentation (MS/MS) by higher-energy collisional
dissociation (HCD) using an NCE of 28 and an AGC target of
1 Â 10
5 in 80 ms at a resolution of 17,500.
The raw data files were processed with the MaxQuant software
(version 1.6.1.0) using the Andromeda search engine to search
MS/MS data against UniProt Mycobacterium smegmatis (strain:
mc
2 155) (6602 entries) database with common contaminants.
Carbamidomethylation of cysteine (C) was set as fixed modification
and oxidation of methionine and NQ acetylation were defined as
variable modifications. Trypsin/P was selected as a protease with
2 missed cleavages allowed and at least two peptides were required
for LFQ quantitation. Precursor mass tolerance was 4.5 ppm and
MS/MS mass tolerance was 20 ppm for HCD fragmentation data
analysis. The false discovery rate (FDR) for peptides and proteins
was set to 1%. For all other parameters, the default settings were
used. Downstream statistical analysis was performed with Perseus
(1.6.10.45).
3.9 Protein
Identification and Data
Handling
Following MaxQuant-based protein identification and quantification, statistical analysis using Perseus software [7, 8] version
1.6.10.45. Protein identifications were filtered to only consider
proteins that had been identified with at least 2 unique peptides.
Reverse contaminants were also filtered out together with potential
contaminants. Single colonies were compared using analysis of
variance test (ANOVA) with p ¼ 0.05, FDR ¼ 0.05.
4 Results
Following MaxQuant-based spectral matching, 52,520 MS/MS
spectra were identified across all replicates. Cumulatively, 7805
unique peptides and 1387 unique proteins were identified at a
0.9% and 0.2% false discovery rate, respectively (Table 1). Following filtering, data distribution was normal (Fig. 1) and reproducibility was high (>94.7%) between replicates (Fig. 2), indicating
that the method detailed herein is highly reproducible. All raw and
spectral search output data were deposited to ProteomeXchange
(6) and are freely available under the identifier PXD018168.
Mass Spectrometry-Based Analysis of Mycobacterial Single-Colony Proteome
185