Computational protocol: Proteoform-Specific Insights into Cellular Proteome Regulation*

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Protocol publication

[…] Data from bottom-up MS analyses of protein OGE separations of hRSV-infected and uninfected A549 cells were acquired and processed as reported in detail previously (). The data set can be downloaded from the MassIVE repository (massive.ucsd.edu/ProteoSAFe/datasets.jsp) with identifier MSV000079453.In brief, data from five independent matched replicate sets of uninfected and hRSV-infected type II alveolar lung epithelial A549 cells were used (). Infection of A549 cells was at a multiplicity of infection (MOI) of three plaque forming units (pfu)/cell using a recombinant clone of hRSV containing the A2 genomic sequence (, ). A549 cells used in this study were verified as Mycoplasma free. Each of the ten samples was lysed 24 h postinfection and individually subjected to protein OGE using a 24 cm nonlinear pH gradient of 3–11. The resultant 24 fractions were individually subjected to tryptic digestion followed by capillary HPLC interfaced with a hybrid linear-ion-trap (LTQ)/OrbitrapXL mass spectrometer.As previously reported (), the 240 .RAW files (Xcalibur, ThremoFisher Scientific) resulting from MS analysis of the 24 fractions from the ten protein OGE separations were processed using MaxQuant 1.3.0.5 () (see () for the full set of MaxQuant parameters). Andromeda () was used to search the complete proteome for Homo sapiens and hRSV A2 strains (87,636 canonical and isoform sequences downloaded from www.uniprot.org on 21 February 2013). A posterior error probability threshold of 0.05 and a peptide level false discovery rate threshold of 0.001 were applied to accept confidently identified PSMs. A protein level false discovery rate of 0.01 was applied. Peptide and protein identifications for each fraction in each protein OGE separation were reported separately (i.e. fractions belonging the same protein OGE separation were not combined). The MaxQuant assembled protein groups were collapsed such that protein sequences arising from the same gene were reported as a single protein group. UniProt accession numbers identified only by peptides matching more than one protein group were excluded from the analysis. Following this procedure, there was no overlap between the protein groups in terms of peptide sequences, UniProt accession numbers or gene names assigned to the protein groups.In the current study, the protein groups reported previously () were further processed using sequence annotations from the UniProt Knowledgebase (UniProtKB). The UniProtKB sequence status and molecule processing annotations were extracted for all UniProt accession numbers assigned to the protein groups (the complete UniProtKB entries were downloaded from www.uniprot.org on 21 February 2013). The UniProtKB sequence status annotation was used to identify UniProt accession numbers with incomplete protein sequences. That is, UniProt accession numbers with sequence status of “fragment” are incomplete protein sequences because of uncertainty in the gene model (e.g. no start and/or stop codon). UniProt accession numbers annotated as fragments were excluded from subsequent analyses. The UniProtKB molecule processing annotation was used to identify processed forms of the protein sequence (e.g. the extent of the protein sequence after the removal of a signal or transit peptide or other cleavage events). All annotated forms of the protein sequence (precursor and processed) were retained for subsequent analyses. Therefore, taking the UniProtKB annotations into account, the final protein groups examined in the current study consisted of an exhaustive list of UniProt accession numbers that matched at least one PSM and included all processed forms of the protein sequence and excluded all protein sequences annotated as fragments. Finally, for each confidently identified peptide, the corresponding UniProt accession numbers, protein group identifier, protein OGE experiment, fraction number and the number of PSMs were extracted from the MaxQuant results file “evidence.txt.” [...] Approximately 33 μl (∼11% of the total fraction volume) aliquots of hRSV-infected and uninfected protein OGE fractions five, six, and seven were combined as two separate pools prior to reduction by addition of 1 m DTT to a final concentration of 10 mm and incubation for 16 h at 4 °C and 2 h at 22 °C. The samples were alkylated and concentrated as described previously (). Approximately one third of the combined and concentrated fractions (five, six, and seven) from hRSV-infected and uninfected samples, described above, were separated by 1D-SDS-PAGE using a 4–20% mini-protean TGX precast polyacrylamide gel (Bio-Rad) according to manufacturer's instructions. The SDS-PAGE lanes of the hRSV-infected and uninfected samples were separately excised and each lane sliced into 52 bands and subjected to in-gel digestion described previously (, ). After overnight incubation of the gel slices with trypsin solution, peptides from the gel pieces were extracted three times for 45min at 37 °C with 40 μl of 0.1% (v/v) TFA and final extraction was performed in 5% (v/v) TFA in 50% (v/v) aqueous ACN. The trypsin supernatant and extracts were pooled and completely dried. In addition, ∼15 μg of protein from one of the whole cell lysate sets used for 1D-Western blotting were subjected to 1D-SDS-PAGE and subsequent steps described above. Resultant digests were reconstituted in 10 μl of 0.1% (v/v) TFA in 2%(v/v) aqueous ACN and 5 μl was subjected to nanoUltraHighPressure Chromatography -MS/MS analysis using a Waters (Milford, MA) NanoAcquity UltraHighPressure chromatography system interfaced to an LTQ-Orbitrap VelosPro hybrid mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) as described previously (, ). The peptides were separated at 35 °C using a sequence of linear gradients: to 27% B over 45 min; to 40% B over 3 min; and, to 95% B over 4 min followed by 95% B for 5 min. The MS/MS data were processed using Proteome Discoverer (version 1.4.1.14; Thermo Fisher Scientific), and searched with Sequest HT () against the same database as the protein OGE data (). Enzymatic cleavage was set to tryptic (allowing a maximum of two missed cleavages) and carbamidomethylation of cysteine was specified as a fixed modification. Deamidation of asparagine/glutamine and methionine oxidation were specified as variable modifications. Fragment ion and precursor ion mass tolerances were set to 0.8 Da and 20 ppm, respectively. Validation of PSMs was performed using Percolator () with a q-value threshold of 0.01. A minimum of two peptides per protein was required for identification and ambiguous protein identifications were reported as protein groups. […]

Pipeline specifications

Software tools Proteome Discoverer, Comet, Percolator
Application MS-based untargeted proteomics
Organisms Human orthopneumovirus, Viruses
Diseases Infection