DOI: 10.5061/dryad.qbzkh18vz
title: A simplified method for comprehensive capture of the Staphylococcus aureus proteome: S. aureus proteome data table

description: Staphylococcus aureus is a major human pathogen causing myriad infections
 in both community and healthcare settings. Although well studied, a
 comprehensive exploration of its dynamic and adaptive proteome is still
 somewhat lacking. Herein, we employed streamlined liquid- and gas-phase
 fractionation with PASEF analysis on a TIMS-TOF instrument to expand
 coverage and explore the S. aureus dark proteome. In so doing, we captured
 the most comprehensive S. aureus proteome to date, totaling 2,231 proteins
 (85.6% coverage), using a significantly simplified process that
 demonstrated high reproducibility with minimal input material. We then
 showcase application of this library for differential expression profiling
 by investigating temporal dynamics of the S. aureus proteome. This
 revealed alterations in metabolic processes, ATP production, RNA
 processing, and stress-response proteins as cultures progressed to
 stationary growth. Notably, a significant portion of the library (94%) and
 proteome (80.5%) was identified by this single-shot, DIA-based analysis.
 Overall, our study shines new light on the unexplored S. aureus proteome,
 generating a valuable new resource to facilitate further study of this
 dangerous pathogen.
description: # A simplified method for comprehensive capture of the Staphylococcus
 aureus proteome: S. aureus proteome data table Dataset DOI:
 [10.5061/dryad.qbzkh18vz](10.5061/dryad.qbzkh18vz) **Data set overview**:
 This dataset contains the data obtained from characterizing the
 *Staphylococcus aureus* LAC USA300 proteome using a simplified yet
 high-throughput methodology for MS/MS capture. This method uses TIMS
 fractionation as well as a simplified liquid-phase fractionation on a
 TIMS-TOF instrument designed to enhance proteome capture while minimizing
 sample processing and hands-on experimental time. We performed statistical
 analysis and quality assessment of this DDA-PASEF library of proteins and
 peptides. Following generation of this comprehensive bacterial proteome,
 we then utilized this resource to perform differential expression
 profiling of various growth phases of *S. aureus*, implementing a
 DIA-single shot approach. We characterized the secreted and cellular
 fractions of mid exponential phase, entry into stationary phase and end
 stationary phase, cultured at 3 hours, 6 hours and 16 hours, respectively,
 also represented in this dataset. **CSV file names correspond to each
 table within the S. aureus proteome
 datatable**: **S._aureus_Proteome_Data_Table_FINAL.xlsx** **Files**
 Legend.csv: This file contains the legend of the *S. aureus* proteome data
 table. A description of the table is listed alongside the table name and
 the Tab of excel spreadsheet it is found on. Number of variables: 3 Number
 of header rows:1 Number of rows: 7 Variable list: *Tab number: (text)
 Excel tab number of each data table. *Legend/Table Number: (alphanumeric)
 legend and table designation of each dataset. *Description: (text)
 Description of each dataset. Data type: alphanumeric, text S.
 aureus_Proteins.csv: This file contains summary data for each identified
 protein from Staphylococcus aureus LAC USA300 strain. Each protein entry
 includes details such as gene name, protein length, organism, protein
 coverage, peptide counts, spectral counts, and intensity. Raw mass
 spectrometry data were searched and processed using FragPipe (v.19.1) with
 the DIA_SpecLib_Quant workflow. MSBooster, Percolator, and ProteinProphet
 were the tools used in FragPipe to enhance confidence in protein and
 peptide identifications. A 1% false discovery rate (FDR) cutoff was used
 when performing spectral assignments. Number of variables: 23 Number of
 header rows: 1 Number of rows: 2232 Variable list: *Protein:
 (alphanumeric) UniProt accession number and identifier for the protein.
 *Protein ID: (alphanumeric) Uniprot ID accession number. *Entry Name:
 (alphanumeric) UniProt entry name. *Gene: (alphanumeric) Gene symbol.
 *Length: (numeric) Number of amino acids in the protein. *Organism: (text)
 Full taxonomic description of the organism. *Protein Description: (text)
 Functional annotation of the protein. *Protein Existence: (text) Evidence
 level for protein existence. *Coverage: (numeric) Percentage of protein
 sequence covered by identified peptides. *Protein Probability: (numeric)
 Probability score for protein identification. *Top Peptide Probability:
 (numeric) Highest probability among the identified peptides. *Total
 Peptides: (numeric) Total number of peptides identified for the protein.
 *Unique Peptides: (numeric) Number of unique peptides for the protein.
 *Razor Peptides: (numeric) Number of peptides shared between proteins but
 assigned to the most probable one. *Total Spectral Count: (numeric) Total
 MS/MS spectra matched to the protein. *Unique Spectral Count: (numeric)
 Spectral count from unique peptides. *Razor Spectral Count: (numeric)
 Spectral count from razor peptides. *Razor Assigned Modifications:
 (numeric): post-translational modifications assigned to razor peptides. M
 Methionine Oxidation, Carbamidomethyl of Cysteine, N-terminal
 modification. *Razor Observed Modifications: (numeric): post-translational
 modifications observed to razor peptides Indistinguishable Proteins:
 (alphanumeric): M=Methionine Oxidation, C=Carbamidomethyl of Cysteine,
 N-term=N-terminal modification. Data type: alphanumeric, numeric, text  
 S. aureus_Peptides.csv: This file contains peptide-level mass spectrometry
 data for the corresponding protein library identified in Table S1 for the
 Staphylococcus aureus LAC USA300 strain. Each row represents a peptide
 along with its associated metadata, including sequence, modifications,
 protein origin, spectral data, and protein description. Both assigned and
 observed post-translational modifications (PTMs) are included, such as
 methionine oxidation and carbamidomethylation, and N-terminal
 modifications. Number of variables:17 Number of header rows: 1 Number of
 rows: 37340 Variable list: *Peptide: (alphanumeric) Amino acid sequence of
 the identified peptide. *Prev AA: (character) Amino acid that precedes the
 peptide in the protein sequence. *Next AA: (character) Amino acid that
 follows the peptide in the protein sequence. *Peptide Length: (numeric)
 Number of amino acid residues in the captured peptide. *Charges: (numeric
 list) Observed charge states of the peptide during MS analysis.
 *Probability: (numeric) Confidence score of the peptide-spectrum match
 (PSM). *Spectral Count: (numeric) Number of spectra corresponding to this
 peptide. *Intensity: (numeric) Measured peptide intensity. *Assigned
 Modifications: (text) Modifications assigned to this peptide. M Methionine
 Oxidation, Carbamidomethyl of Cysteine, N-terminal modification. *Observed
 Modifications: (text) Modifications directly identified from mass spectral
 data. M Methionine Oxidation, Carbamidomethyl of Cysteine, N-terminal
 modification. *Protein: (alphanumeric) Uniprot accession ID number and
 identifier for the matched protein. *Protein ID: (alphanumeric) Uniprot
 accession ID number for the matched protein *Entry Name: (alphanumeric)
 UniProt entry name of the matched protein. *Gene: (alphanumeric) Gene
 symbol of the matched protein. *Protein Description: (text) Functional
 annotation or description of the matched protein. *Mapped Genes: (text)
 Genes mapped from the matched protein. *Mapped Proteins: (text) Functional
 protein group or family associated with the matched protein. Data type:
 alphanumeric, numeric, character, text. DIALib-QC_Quality_Assessment.csv:
 This file contains information from the quality assessment using the
 DIALib-QC tool (publicly available) of the generated Staphylococcus aureus
 DDA-PASEF library of proteins and peptides. Headings are labelled with the
 parameters that were assessed using this tool. Number of variables:15
 Number of header rows: 1 Number of rows:3 Variable list with abbreviations
 in the row below (highlighted in bold): *Name of library file being
 analyzed (library): (text) Name assigned to the spectral library file.
 *Library format (OpenSWATH, Peakview, or Spectronaut) (format): (text)
 Format used for the library. *Number of peptide ions (i.e., precursor,
 sequence + modifications + charge) (pepions): (numeric) Total count of
 peptide ions including sequence, modifications, and charge states. *Number
 of fragments (fragment ions) in library (Fragments): (numeric) Total
 number of fragment ions included in the library. *Percentage of
 proteotypic peptide ions (not shared) (ptp_percent): (numeric) Percent of
 peptide ions that are shared between proteins. *Percentage of shared
 peptide ions (shared_percent): (numeric) Percent of total peptide ions
 that are shared. *Number of shared peptide ions (shared_pepions):
 (numeric) Count of shared peptide ions in the library. *Number of distinct
 peptide sequences (peptides): (numeric) Unique peptide sequences without
 modification. *Number of distinct modified peptides (sequences +
 modifications) (mod_peps): (numeric) Unique peptide sequences including
 modifications. *Percentage of distinct modified peptides with mass
 modification (mod_percent): (numeric) Proportion of peptides with
 mass-modifying PTMs. *Number of mass-modified amino acids (total_mods):
 (numeric) Total number of individual amino acids with mass modifications.
 *Percentage of charge 2 precursors (chg_2): (numeric) Proportion of
 precursors with a +2 charge state. *Percentage of charge 3 precursors
 (chg_3): (numeric) Proportion of precursors with a +3 charge state.
 *Minimum precursor m/z (mass/charge) in library (precursor_min): (numeric)
 Lowest mass-to-charge ratio for a precursor ion in the library. *Maximum
 precursor m/z in library (precursor_max): (numeric) Highest mass-to-charge
 ratio for a precursor ion in the library. *Average peptide length
 (avg_len): (numeric) Mean number of amino acids per peptide. *Average
 number of fragments per assay (precursor) (avg_num_frags): (numeric) Mean
 number of fragment ions associated with each precursor. *Average fragment
 sequence length (avg_frag_len): (numeric) Mean length of the fragment ion
 sequences. *Percentage of assays with 5 or fewer transitions (short_perc):
 (numeric) Fraction of assays that include five or fewer transitions
 (fragment ions). *Percentage of fragment m/z above precursor m/z
 (fragment_above_precursor): (numeric) Percentage of fragments with a
 higher m/z than the precursor ion. *Percentage of y ions (y_perc):
 (numeric) Proportion of fragments classified as y-type ions. *Percentage
 of b ions (b_perc): (numeric) Proportion of fragments classified as b-type
 ions. *Percentage of y ions considering only the top 6 fragments per assay
 (t6_y_perc): (numeric) Percentage of y-type ions among the top 6 most
 intense fragments per assay. *Average intensity (avg_intensity): (numeric)
 Mean intensity of the peptide fragments (NaN if not available). *Minimum
 retention time (RT) in library (rt_min): (numeric) Shortest retention time
 of any peptide in the library. Time = seconds (s) *Maximum RT in library
 (rt_max): (numeric) Longest retention time of any peptide in the library.
 Time = seconds (s) Median RT in library (rt_med): (numeric) Median
 retention time of peptides. Time = seconds (s) r-squared value of fit
 between RT of +2 and +3 charge states for the same modified peptide
 (rt_rsq): (numeric) R¬≤ correlation for RT alignment across charge states.
 Percentage of +2/+3 charge pairs of the same modified peptide within 5 RT
 units of each other(rt_five): (numeric) Consistency of RT for peptides
 across charge states. Number of iRT peptides in library (n_irt): (numeric)
 Count of indexed retention time (iRT) standards present in the library.  
 Data type: numeric, text *Differential_Expression_Analysis_Secreted.csv:
 This file contains quantitative data for DIA single-shot global analysis
 of Staphylococcus aureus LAC USA300 proteins identified across various
 time points (3-hour, 6-hour, and 16-hour conditions). Here we are
 characterizing the different growth phases of *S. aureus *including mid
 exponential phase (3h), entry into stationary phase (6h), and end
 stationary phase (16h) using the DDA-PASEF spectral library of the S.
 aureus proteome identified in Tables S1 and S2. This file is the analysis
 of the secreted fraction of these growth timepoints. Protein groups are
 identified with associated gene annotations and descriptions at each time
 point. We also include replicate intensity values and the corresponding
 log2-transformed values. Original intensity values are also outlined
 before imputation and normalization. Ontological categorizations are
 provided, and statistical comparisons between conditions are made to
 evaluate changes in protein abundance. Proteins in purple text represent
 those where at least one of the two protein groups that were compared has
 2 or fewer MS detected values prior to imputation. Proteins highlighted in
 red indicate that imputation occurred on all four reps for that protein,
 and there was no original MS detection. Number of variables: 83 Number of
 header rows: 1 Number of rows: 501 Variable list: *Protein_Group:
 (alphanumeric) Uniprot group identifier for clustered proteins.
 *Protein_Ids: (alphanumeric) Uniprot Protein accession(s) in the group.
 *Protein_Ids_1: (alphanumeric) Primary protein Uniprot accession in the
 group *Protein_Names: (text): Uniprot identifier for the primary protein
 *Genes: (alphanumeric) Gene symbol(s) for all proteins in the group.
 *Genes_1: (alphanumeric) Gene symbol for the primary protein.
 *First_Protein_Description: (text) Functional annotation of the first
 protein in the group. *Sec Emp 16h v 6h: (numeric): Ratio comparison
 between 16-hour and 6-hour conditions. *Sec Emp 16h v 3h: (numeric) Ratio
 comparison between 16-hour and 3-hour conditions. *Sec Emp 6h v 3h:
 (numeric) Ratio comparison between the 6-hour and 3-hour conditions.
 Replicate Intensities (numeric): *3hr1Sec_LS25 to 3hr4Sec_LS28: Protein
 intensities for 3-hour secretory replicates. *6hr1Sec_LS29 to
 6hr4Sec_LS32: Protein intensities for 6-hour secretory replicates.
 *16hr1Sec_LS33 to 16hr4Sec_LS36: Protein intensities for 16-hour secretory
 replicates. Log2 of Replicate Intensities (numeric): *Log2 3hr1Sec_LS25 to
 3hr4Sec_LS28: Log 2 Protein intensities for 3-hour secretory replicates.
 *Log2 6hr1Sec_LS29 to 6hr4Sec_LS32: Log 2 Protein intensities for 6-hour
 secretory replicates. *Log2 16hr1Sec_LS33 to 16hr4Sec_LS36: Log 2 Protein
 intensities for 16-hour secretory replicates. *ANOVA Significant: (text)
  A one-way ANOVA was performed, followed by a post hoc Tukey’s HSD test
 (FDR&lt;0.05) to determine the significance of differential expression
 when comparing the proteomes at different time points (16vs3 h, 16vs6 h,
 and 6 vs. 3 h) for the secretory fraction. *Significant pairs:
 (alphanumeric) pair-wise comparisons for the secretory fraction timepoints
 that are ANOVA significant *Cluster: (alphanumeric)Cluster designation
 based on hierarchical clustering *GOBP name: (text) name of Gene Ontology
 Biological Process for all proteins in the group *GOMF name: (text) name
 of Gene Ontology Molecular Function for all proteins in the group *GOCC
 name: (text) name of Gene Ontology Cellular Component for all proteins in
 the group *KEGG name: (text) name from the Kyoto Encyclopedia of Genes and
 Genomes *GOBP name_: (text) name of Gene Ontology Biological Process for
 the main protein *GOMF name_:(text)  name of Gene Ontology Molecular
 Function for the main protein *GOCC name_:(text)  name of Gene Ontology
 Cellular Component for the main protein *Uniprot function: (text) function
 assigned to the protein from the Uniprot database *Uniprot subcellular
 location: (text)  subcellular location assigned to the protein from the
 Uniprot database *“-Log ANOVA p value”: (numeric) -Log of the ANOVA p
 value *Protein_Group: (alphanumeric) Group identifier for the proteins in
 the group. *Protein_Ids: (alphanumeric) Uniprot ID(s) for the proteins in
 the group *Protein Ids_1: (alphanumeric) UniProt ID for the primary
 protein *Protein_Names: (alphanumeric) Uniprot identifiers for the
 protein(s) in the group *Genes: (text) Gene name for the proteins in the
 group Genes_1 (text): Gene name for the primary protein
 *First_Protein_Description: (text) Functional annotation of the primary
 protein. Protein Intensities after Imputation and Normalization: *Original
 Cell 3hr1_LS1+13 to Original Cell 3hr4_LS4+16 : (numeric) Protein
 intensity values from the secreted fraction of each 3-hour replicate
 *Original Cell 6hr1_LS5+17 to Original Cell 6hr4_LS8+20: (numeric) Protein
 intensity values from the secreted fraction of each 6-hour replicate
 *Original Cell 16hr1_LS9+21 to Original Cell 16hr4_LS12+24: (numeric)
 Protein intensity values from the secreted fraction of each 3-hour
 replicate Protein Intensities prior to Imputation and normalization:
 *Original 3hr1Sec_LS25 to Original 3hr2Sec_LS26: (numeric): LFQ
 intensities for the secreted fraction of the 3-hour replicated prior to
 imputation and normalization *rep (numeric): number of replicates with
 identified intensities for the 3-hour replicates (preceding) Original
 6hr1Sec_LS29 to Original 6hr3Sec_LS31: (numeric) LFQ intensities for the
 secreted fraction of the 6-hour replicated prior to imputation and
 normalization *rep (numeric): number of replicates with identified
 intensities for the 6-hour replicates (preceding) *Original 16hr2Sec_LS34
 to Original 16hr3Sec_LS35: (numeric): LFQ intensities for the secreted
 fraction of the 16 hour replicated prior to imputation and normalization
 *rep (numeric): number of replicates with identified intensities for the
 16-hour replicates (preceding) *Genes_1_end: (alphanumeric) Gene names for
 the primary protein *Significant pairs: (alphanumeric): Pair wise
 comparisons of timepoints that are significant  Abbreviations: Sec=
 secreted GOBP = Gene Ontology Biological Process GOMP= Gene Ontology
 Molecular Function GOCC= Gene Ontology Cellular Component Data type:
 alphanumeric, numeric, text  
 *Differential_Expression_Analysis_Cellular.csv: This file contains
 quantitative data for DIA single-shot global analysis of Staphylococcus
 aureus LAC USA300 proteins identified across various time points (3-hour,
 6-hour, and 16-hour conditions). Here we are characterizing the different
 growth phases of *S. aureus *including mid exponential phase (3h), entry
 into stationary phase (6h), and end stationary phase (16h) using the
 DDA-PASEF spectral library of the S. aureus proteome identified in Tables
 S1 and S2. This file is the analysis of the cellular of these growth
 timepoints. Protein groups are identified with associated gene annotations
 and descriptions at each time point. We also include replicate intensity
 values and the corresponding log2-transformed values. Original intensity
 values are also outlined prior to imputation and normalization.
 Ontological categorizations are provided, and statistical comparisons
 between conditions are made to evaluate changes in protein abundance.
 Proteins in purple text represent those where at least one of the two
 protein groups that were compared has 2 or fewer MS detected values prior
 to imputation. Proteins highlighted in red indicate that imputation
 occurred on all four reps for that protein, and there was no original MS
 detection.  Number of variables: 83 Number of header rows: 1 Number of
 rows: 709 Variable list: *Protein_Group: (alphanumeric) Uniprot group
 identifier for clustered proteins. *Protein_Ids: (alphanumeric) Uniprot
 protein accession(s) in the group. *Protein_IDs_1: (alphanumeric) Primary
 protein Uniprot accession in the group *Protein_Names: (text): Uniprot
 identifier for the primary protein *Genes: (alphanumeric) Gene symbol(s)
 for all proteins in the group. *Genes_1: (alphanumeric) Gene symbol for
 the primary protein. *First_Protein_Description: (text) Functional
 annotation of the first protein in the group. *Cell Emp 16h vs 6h:
 (numeric) Ratio comparison between 16-hour and 6-hour conditions. *Cell
 Emp 16h vs 3h: (numeric) Ratio comparison between 16-hour and 3-hour
 conditions. *Cell Emp 6h vs 3h: (numeric) Ratio comparison between the
 6-hour and 3-hour conditions. Replicate Intensities (numeric): *Cell
 3hr1_LS1+13 to Cell 3hr4_LS4+16: Protein intensities for 3-hour cellular
 replicates. *Cell 6hr1_LS5+17 to Cell 6hr4_LS8+20: Protein intensities for
 6-hour cellular replicates. *Cell 16hr1_LS9+21 to Cell 16hr4_LS12+24:
 Protein intensities for 16-hour cellular replicates. Log2 of Replicate
 Intensities (numeric): *Log2 Cell 3hr1_LS1+13 to Log2 Cell 3hr4_LS4+16:
 Log 2 Protein intensities for 3-hour cellular replicates. *Log2 Cell
 6hr1_LS5+17 to Log2 Cell 6hr4_LS8+20: Log 2 Protein intensities for 6-hour
 cellular replicates. *Log2 Cell 16hr1_LS9+21 to Log2 Cell 16hr4_LS12+24:
 Log 2 Protein intensities for 16-hour cellular replicates. *ANOVA
 Significant: (text) A one-way ANOVA was performed, followed by a post hoc
 Tukey’s HSD test (FDR&lt;0.05) to determine the significance of
 differential expression when comparing the proteomes at different time
 points (16vs3 h, 16 vs. 6 h, and 6vs3h) for the cellular fraction.
 *Significant pairs: (alphanumeric) pair-wise comparisons for the cellular
 fraction timepoints that are ANOVA significant *Cluster: (alphanumeric)
 Cluster designation based on hierarchical clustering *GOBP name: (text)
 name of Gene Ontology Biological Process for all proteins in the group
 *GOMF name: (text) name of Gene Ontology Molecular Function for all
 proteins in the group *GOCC name: (text) name of Gene Ontology Cellular
 Component for all proteins in the group *KEGG name: (text) name from the
 Kyoto Encyclopedia of Genes and Genomes *GOBP name_: (text) name of Gene
 Ontology Biological Process for the main protein *GOMF name_:(text) name
 of Gene Ontology Molecular Function for the main protein *GOCC
 name_:(text) name of Gene Ontology Cellular Component for the main protein
 *Uniprot function: (text) function assigned to the protein from the
 Uniprot database *Uniprot subcellular location: (text) subcellular
 location assigned to the protein from the Uniprot database *“-Log ANOVA p
 value”: (numeric) -Log of the ANOVA p value *Protein_Group: (alphanumeric)
 Group identifier for the proteins in the group. *Protein_Ids:
 (alphanumeric) Uniprot ID(s) for the proteins in the group *Protein Ids_1:
 (alphanumeric) UniProt ID for the primary protein *Protein_Names:
 (alphanumeric) Uniprot identifiers for the protein(s) in the group *Genes:
 (text) Gene name for the proteins in the group *Genes_1 (text): Gene name
 for the primary protein *First_Protein_Description: (text) Functional
 annotation of the primary protein. Protein Intensities prior to Imputation
 and normalization: *Original Cell 3hr1_LS1+13 to Original Cell
 3hr4_LS4+16: (numeric): LFQ intensities for the cellular fraction of the
 3-hour replicated prior to imputation and normalization *rep (numeric):
 number of replicates with identified intensities for the 3-hour replicates
 (preceding) *Original Cell 6hr1_LS5+17 to Original Cell 6hr4_LS8+20:
 (numeric) LFQ intensities for the cellular fraction of the 6-hour
 replicated prior to imputation and normalization *rep (numeric): number of
 replicates with identified intensities for the 6-hour replicates
 (preceding) *Original Cell 16hr1_LS9+21 to Original Cell 16hr4_LS12+24:
 (numeric): LFQ intensities for the cellular fraction of the 16-hour
 replicated prior to imputation and normalization *rep (numeric): number of
 replicates with identified intensities for the 16-hour replicates
 (preceding) Raw Protein Intensities: *Original 3hr1Sec_LS25 to Original
 3hr4Sec_LS28: (numeric) Protein intensity values from the cellular
 fraction of each 3-hour replicate *Original 6hr1Sec_LS29 + Original
 6hr4Sec_LS32: (numeric) Protein intensity values from the cellular
 fraction of each 6-hour replicate *Original 16hr2Sec_LS34 to Original
 16hr3Sec_LS35: (numeric) Protein intensity values from the cellular
  fraction of each 3-hour replicate *Genes_1_end: (alphanumeric) Gene names
 for the primary protein *Significant pairs: (alphanumeric): Pairwise
 comparisons of timepoints that are significant Abbreviations: Cell=
 Cellular GOBP = Gene Ontology Biological Process GOMP = Gene Ontology
 Molecular Function GOCC = Gene Ontology Cellular Component **Version
 Changes:**  **02-22-25**: Files were given more descriptive names as per
 the journals request. Changes to the data were not made.