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An equal volume of recovered sample was analyzed by LC-MS/MS as follows. Chromatography was performed on a Thermo Vanquish Neo UHPLC in “heated trap-and-elute, backward flush” mode. Peptides were desalted and concentrated on a PepMap Neo trap column (300 uM i.d. x 5 mm, 5 µm C18, 100 A u-Precolumn, Thermo Scientific) at a flow rate of 5 µL min-1. Sample separation was performed on a Aurora Ultimate Column (IonOpticks) with a flow rate of ~350 nL min-1 over a 60 min reverse phase gradient followed by a column/trap wash at 80% ACN for 10 min. Eluted peptides were analyzed using a Thermo Scientific Orbitrap Exploris 480 mass spectrometer with a FAIMS pro Duo interface installed, which was directly coupled to the UHPLC through a Flex Ion source (Thermo Scientific). Data Independent Acquisition (DIA) was performed using Xcalibur 4.0 software in positive ion mode with a spray voltage of 2.0 kV, a capillary temperature of 280 C, an RF of 45, FAIMS compensation voltage of -45, and a total carrier gas flow of 4.2 l/min. A full scan was acquired at a resolution of 60,000, a scan range o 400-900, a normalized automatic gain control (AGC) target % of 225, and absolute AGC value of 2.25e6, and maximum inject time set to auto. Forty-two DIA acquisition windows of 12m/z with 1Da overlap were measured at a resolution of 30,000 with a normalized AGC Target % of 800, an absolute AGC of of 8e5, and auto maximum ion time, over a precursor mass range of 400-900 m/z. A scan range 145-1450 m/z and normalized collision energy of 29 were used."],"repository":["Pride"],"quantification_method":["Not available"],"modification":[""],"data_protocol":["Raw data were analyzed using Spectronaut version 19 (Biognosis). Spectra were searched, using the Pulsar search engine against the P. aeruginosa UCBPP-PA14 (Genome assembly ASM1462v1) and Staphylococcus aureus JE2 (Genome assembly ASM208552v1) reference genomes downloaded from NCBI. The spectra search was performed on “direct DIA” mode. Carbamidomethyl cysteine was set as a fixed modification while methionine oxidation and protein N-terminal acetylation were set as variable modifications. Digestion parameters were set to “specific” and “Trypsin/P,LysC”. Up to two missed cleavages were allowed. A false discovery rate, calculated using a “KR” decoy generation rule, of less than 0.01 at the peptide spectral match, peptide, and protein group identification level was required. Filtering of Mass Spectrometry data and identification of unique PTMs. To correct for background signal in our acylation datasets, protein-level quantifications from the DMSO control were compared to those observed under each acylation condition. For each protein, the average log2 fold-change, along with the corresponding P-value and Q-value, was calculated by statistical comparison against the DMSO control. Only proteins with statistically significant enrichment (above the DMSO background) were retained for inclusion in the final acylation data set (Figure S1-S5). Proteomics data from each experimental condition were processed to retain only protein groups with a positive fold-change with contaminant proteins removed. Protein group identifiers (NCBI) were used for comparison of acetylation, propionylation, and butyrylation data sets. To determine unique protein groups for each PTM, the processed csv files for each condition were loaded into Python (version 3.7.6) using pandas (version 0.24.2). For each file, protein group identifiers present in the test condition but absent from the other data sets were identified as unique. The unique rows for each sample were exported to separate csv files for downstream analysis. Proteins shared between multiple or all datasets were found be taking the intersection of sets and relevant data exported"],"omics_type":["Proteomics"],"labhead":["Justin Walley"],"instrument_platform":[""],"submission_type":["PARTIAL"],"labhead_affiliation":["Department of Plant Pathology, Entomology, and Microbiology Iowa State University"],"species":["Pseudomonas Aeruginosa (strain Ucbpp-pa14)","Staphylococcus Aureus"],"publication":["41697727 Monacchio HN, Shah RS, Montes CF, Wang GZ, Walley JW, VanDrisse CM. Bioorthogonal Click Chemistry for Antibody-Free Profiling of Acetylation, Propionylation, and Butyrylation in &lt;i&gt;Pseudomonas aeruginosa&lt;/i&gt; and Methicillin-Resistant &lt;i&gt;Staphylococcus aureus&lt;/i&gt;. ACS Infect Dis. 2026 12(3):1142-1154 10.1021/acsinfecdis.5c00985"],"submitter_mail":["chrisfm@iastate.edu"],"submitter_affiliation":["Iowa State University"],"submitter_country":["United States"],"pubmed_abstract":["Lysine acylation is a posttranslational modification (PTM) conserved in all domains of life and is essential for regulating diverse biological processes. Traditional methods for investigating acylation rely on anti-acyl-lysine antibodies, which are costly and time-consuming and often exhibit variable affinity. To remedy these pitfalls, we developed an antibody-free method for bacterial acylome enrichment using bioorthogonal click chemistry coupled with tandem mass spectrometry. We applied this approach to the pathogens <i>Pseudomonas aeruginosa</i> and methicillin-resistant <i>Staphylococcus aureus</i> (MRSA) to explore the biological significance of acylation in each organism. We characterized the acetylome, propionylome, and butyrylome in <i>P. aeruginosa</i> UCBPP-PA14 and the acetylome and propionylome in MRSA. Comparative analyses revealed unique PTM dynamics showing that acylation regulated a wide range of cellular functions, including metabolism, antibiotic resistance, virulence, and stress response. This work establishes the first antibody-free enrichment method for defining bacterial acylomes and provides new insight into global lysine acylation networks in pathogenic bacteria."],"pubmed_title":["Bioorthogonal Click Chemistry for Antibody-Free Profiling of Acetylation, Propionylation, and Butyrylation in &lt;i&gt;Pseudomonas aeruginosa&lt;/i&gt; and Methicillin-Resistant &lt;i&gt;Staphylococcus aureus&lt;/i&gt;."],"pubmed_authors":["Monacchio Haley N HN, Shah Ritika S RS, Montes Christian F CF, Wang Grace Z GZ, Walley Justin W JW, VanDrisse Chelsey M CM"],"additional_accession":[]},"is_claimable":false,"name":"Bioorthogonal Click Chemistry for Antibody-Free Profiling of Acetylation, Propionylation, and Butyrylation in Pseudomonas aeruginosa and Methicillin-Resistant Staphylococcus aureus","description":"Lysine acylation is a post-translational modification (PTM) conserved in all domains of life and is essential for regulating diverse biological processes. Traditional methods for investigating acylation rely on anti-acyl-lysine-antibodies, which are costly, time-consuming, and often exhibit variable affinity. To remedy these pitfalls, we developed an antibody-free method for bacterial acylome enrichment using bioorthogonal click chemistry coupled with tandem mass spectrometry. We applied this approach to the pathogens Pseudomonas aeruginosa and methicillin-resistant Staphylococcus aureus (MRSA) to understand the regulatory role of acylation in each organism. We characterized the acetylome, propionylome and butyrylome in P. aeruginosa UCBPP-PA14 and the acetylome and propionylome in MRSA. Comparative analyses revealed distinct PTM dynamics showing that acylation regulated a wide range of cellular functions, including metabolism, antibiotic resistance, virulence, and stress response. This work establishes the first antibody-free enrichment method for defining bacterial acylomes and provides new insight into global lysine acylation networks in pathogenic bacteria.","dates":{"publication":"2026-06-11","submission":"2026-01-01"},"accession":"PXD072564","cross_references":{"TAXONOMY":["NEWT:330879","NEWT:377960","NEWT:1131","NEWT:35795","NEWT:2042546","NEWT:259447","NEWT:295546","NCBITaxon:2719036","NEWT:1334565","NEWT:625351","NEWT:258594","NCBITaxon:1280","NEWT:112503","NEWT:1129","NEWT:309807","NEWT:59753","NEWT:89184","NEWT:309800","NEWT:281395","NEWT:1211601","NEWT:876138","NEWT:44271","NEWT:193516","NEWT:2315688","NEWT:1073882","NEWT:6819","NEWT:475174","NEWT:111205","NEWT:428386","NEWT:1117","NEWT:498257","NEWT:10036","NEWT:32264","NEWT:1590","NEWT:10034","NEWT:33114","NEWT:1596","NEWT:661410","NEWT:638632","NEWT:224326","NEWT:376619","NCBITaxon:79857","NEWT:1096976","NEWT:3765","NEWT:3767","NEWT:1589","NEWT:135622","NEWT:4615","NEWT:672127","NEWT:35786","NEWT:1580","NEWT:399784","NEWT:96731","NEWT:656064","NEWT:1582","NEWT:3760","NEWT:1584","NEWT:36630","NEWT:1585","NEWT:383379","NEWT:418106","NCBITaxon:12721","NEWT:10029","NEWT:913645","NEWT:2492962","NEWT:641809","NEWT:1392488","NEWT:38815","NEWT:317447","NEWT:4688","NEWT:111225","NEWT:7719","NEWT:5530","NEWT:868565","NEWT:135674","NEWT:79329","NEWT:30069","NEWT:12637","NEWT:59729","NEWT:2164133","NEWT:295105","NEWT:108061","NEWT:60711","NEWT:224308","NEWT:3347","NEWT:49928","NEWT:160621","NEWT:212790","NEWT:110368","NEWT:1310161","NEWT:77133","NEWT:145481","NEWT:1310165","NEWT:29058","NCBITaxon:79824","NEWT:410658","NEWT:5516","NEWT:44688","NEWT:44689","NEWT:498211","NEWT:347256","NEWT:5518","NEWT:398007","NEWT:1527468","NEWT:498217","NEWT:498216","NEWT:11320","NEWT:246196","NEWT:246197","NEWT:145458","NEWT:1310173","NEWT:44685","NEWT:161934","NEWT:156587","NEWT:1148","NEWT:5508","NEWT:3329","NEWT:419481","NEWT:5507","NEWT:410661","NEWT:1140","NCBITaxon:2157","NEWT:2066563","NEWT:1143","NEWT:1287689","NEWT:1094343","NEWT:1462472","NEWT:1336795","NEWT:644042","NEWT:150808","NEWT:1182590","NEWT:3712","NEWT:3711","NEWT:3714","NCBITaxon:37162","NEWT:270643","NEWT:2065263","NEWT:177437","NEWT:10891","NEWT:10418","NEWT:118698","NEWT:1616117","NEWT:118696","NEWT:34865","NEWT:52283","NEWT:28174","NEWT:284812","NEWT:8175","NEWT:43330","NEWT:1603293","NEWT:44664","NEWT:43346","NEWT:3702","NEWT:1245466","NEWT:3704","NEWT:244366","NEWT:1246791","NEWT:176597","NEWT:118694","NEWT:2850","NEWT:118691","NEWT:34871","NEWT:33548","NEWT:52299","NEWT:8165","NEWT:96794","NEWT:3708","NEWT:332648","NEWT:44670","NEWT:926571","NEWT:536231","NEWT:376219","NEWT:219813","NEWT:1510","NEWT:460519","NEWT:1515","NEWT:572307","NEWT:1432138","NEWT:1424507","NEWT:1194599","NEWT:272844","NEWT:1348799","NEWT:33995","NEWT:55784","NEWT:1303443","NEWT:9483","NEWT:485","NEWT:56636","NEWT:2499525","NEWT:2853422","NEWT:487","NEWT:1538553","NEWT:1679718","NEWT:480","NEWT:67767","NEWT:46835","NEWT:109757","NEWT:582580","NEWT:1185650","NEWT:216778","NEWT:294607","NEWT:1502","NEWT:128017","NEWT:376686","NEWT:237610","NEWT:95486","EFO:0001352","NEWT:119562","NEWT:1883446","NEWT:1233435","NEWT:118223","NEWT:11757","NEWT:987059","NEWT:109760","NEWT:29031","NEWT:317047","NEWT:1509","NEWT:491","NEWT:460523","NEWT:45954","NEWT:235443","NEWT:1579","NEWT:1757312","NEWT:85694","NEWT:109786","NEWT:108458","NEWT:5936","NEWT:320637","NEWT:1078283","NEWT:3750","NEWT:983964","NEWT:11706","NEWT:32644","NEWT:527796","NEWT:499175","NEWT:109779","NEWT:3745","NEWT:1715989","NCBITaxon:4751","NEWT:3747","NEWT:1116234","NEWT:1255228","NEWT:410289","NEWT:191701","NEWT:373153","NEWT:1640278","NEWT:472","NEWT:940825","NEWT:29459","NEWT:529507","NEWT:1071661","NEWT:28128","NEWT:470","NEWT:5911","NEWT:748693","NCBITaxon:50557","NEWT:39251","NEWT:29491","NEWT:101841","NEWT:446","NEWT:153481","NEWT:2014887","NEWT:33952","NEWT:11723","NEWT:445","NEWT:153009","NEWT:261756","NEWT:166313","NEWT:63366","NEWT:63367","NEWT:215402","NEWT:1547","NEWT:1290454","NEWT:906914","NEWT:81231006","NEWT:28141","NEWT:9031","NEWT:27292","NEWT:1050722","NEWT:3728","NEWT:108931","NEWT:1293497","NEWT:1055524","NEWT:548","NEWT:150475","NEWT:267872","NEWT:381666","NEWT:172269","NEWT:9534","NEWT:2126348","NEWT:5180","NEWT:256737","NEWT:9541","NEWT:8694","NEWT:33936","NEWT:8692","NEWT:2903","NEWT:185579","NEWT:13076","NEWT:207340","NEWT:1006581","NEWT:8673","NEWT:33940","NEWT:550","NEWT:33941","NEWT:554","NEWT:451516","NEWT:552","NEWT:1325291","NEWT:42410","NEWT:36185","NEWT:1054211","NEWT:1225786","NEWT:771870","NEWT:51338","NEWT:9995","NEWT:575412","NEWT:28112","NEWT:49012","NEWT:6493","NEWT:10832","NEWT:6494","NEWT:6491","NEWT:507601","NEWT:520","NEWT:186441","NEWT:643680","NEWT:214092","NCBITaxon:6157","NEWT:13095","NEWT:162425","NEWT:104105","NEWT:216257","NEWT:9986","NEWT:8654","NEWT:8658","NEWT:1268063","NEWT:8657","NEWT:9983","NEWT:8655","NEWT:5147","NEWT:28108","NEWT:28104","NEWT:407821","NEWT:568703","NCBITaxon:2","NEWT:568708","NEWT:1613","NEWT:986","NEWT:52641","NEWT:28532","NEWT:353152","NEWT:298176","NEWT:353153","NEWT:227377","NEWT:1774284","NEWT:40674","NEWT:1194669","NEWT:51329","NEWT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