MassIVEapplication/xmlftp://massive-ftp.ucsd.edu/v08/MSV000095427/primaryOK200John P. LaCavahttps://massive.ucsd.edu/ProteoSAFe/dataset.jsp?task=e4544446b22e4353b6929d360cbd72b6jlacava@rockefeller.eduMassIVE207MOD:00110 - "A protein modification that effectively converts an L-cysteine residue to L-cysteine methyl disulfide."UNIMOD:35 - "Oxidation or Hydroxylation."ProteomicsOrbitrap Exploris 480Homo Sapiens (ncbitaxon:9606)University of GroningenData-Independent Acquisition (DIA) LC-MS/MS is an attractive partner for co-immunoprecipitation (co-IP) and affinity proteomics in general. Reducing the variability of quantitation by DIA could increase the statistical contrast for detecting specific interactors versus what has been achieved in Data-Dependent Acquisition (DDA). By interrogating affinity proteomes featuring both DDA and DIA experiments, we sought to evaluate the spectral libraries, the missingness of protein quantity tables, and the CV of protein quantities in six studies representing three different instrument manufacturers. We examined four contemporary bioinformatics workflows for DIA: FragPipe, DIA-NN, Spectronaut, and MaxQuant. We determined that (1) identifying spectral libraries directly from DIA experiments works well enough that separate DDA experiments do not produce larger spectral libraries when given equivalent instrument time; (2) experiments involving mock pull-downs or IgG controls may feature such indistinct signals that contemporary software will struggle to quantify them; (3) measured CV values were well controlled by Spectronaut and DIA-NN (and FragPipe, which implements DIA-NN for the quantitation step); and (4) when FragPipe builds spectral libraries and quantifies proteins from DIA experiments rather than performing both operations in DDA experiments, the DIA route results in a larger number of proteins quantified without missing values as well as lower CV for measured protein quantities.<h4>Supplementary information</h4>The online version contains supplementary material available at 10.1007/s42485-024-00166-4.Interrogating data-independent acquisition LC-MS/MS for affinity proteomics.Tabb David L DL, Kaniyar Mohammed Hanzala MH, Bringas Omar G Rosas OGR, Shin Heaji H, Di Stefano Luciano L, Taylor Martin S MS, Xie Shaoshuai S, Yilmaz Omer H OH, LaCava John Jliquid chromatography tandem mass spectroscopy, LC-MS2, LC-MS/MS, liquid chromatography tandem mass spectrometry, LC-MS-MS., LC/MS/MS, LCMSMS, liquid chromatography-tandem mass spectroscopy, DIA, LC-MSMSsodium salt, WASLBP, Eph-like tyrosine kinase 1, IgG Antibody, Co-Immunoprecipitations, Carcinoma Cell, galere, DmelCG10561, Laboratory, IPS, Mus domesticus, leg, Stem Cells, ino1-B, ETK1, Splice Variants, E(sev)3A, Protein Splice, House Mouse, Long Term, Wasbp, Co Immunoprecipitation, Carcinoma Cells, l(1)AA33, DmelCG1242, stc, Gamma Globulin, ips, 1, 2, bis(p-chlorophenyl)acetic acid, Gpi-1, Embryonal Carcinoma Cells, Human embryo kinase, Dmel_CG18042, Effect, TYRO4, MMTS, HSP82, l(2)37Cs, anon-WO0068693, Protein Splice Variant, Man (Taxonomy), Nlk, C2, Co-Immunoprecipitation, EK4, Hsp90, Precipitation, Swiss Mice, isyna1, E(sina)2, Protein Isoform, l(3)j5C2, HSP90, Immune, dTAB2, dTab2, l(1)B2/13.1, DIP, RUN, Run, Teratocarcinoma, methyl methanesulfonothioate, CG7417, cs, Hsp82, house mouse, Gm641, Precipitations, Long-Term Effects, HSP83, NK/GPI, Gpi, F9 Teratocarcinoma Stem Cells, ichthyosis-prematurity syndrome, l(1)LB9, TuC1, anon-37Cd, anon-37Cb, anon-37Cc, WISH, Modern, Cs, mouse, cg7417, Longterm Effect, Teratocarcinoma Stem Cells, CG1242, Isoform, IPS-1, Teratocarcinoma Stem Cell, AA33, anon-37Cs, 83K HSP, en(lz)3C/4C, mOC-X, IgG3, Stem Cell, IgG4, IgG1, IgG2, Clones, APC11, ichthyosis congenita 4, Teratocarcinoma Stem, EPH-like kinase 4, NK|GPI, Mus musculus, DmelCG7417, Org, ORG, IgG, anon-EST:Liang-2.53, mice, Swiss Mouse, Gpi1-r, Gpi1-s, methyl methanethiolsulfonate, hEK4, Gpi1-t, Apc11, human, Clone, domesticus, LMG, IgG2A, IgG2B, GT, Cells, DmelCG34440, HEK, ACSVL4, Embryonal, SPIN90, Mouse, ino1-a, Mage-d2, DmelCG14965, dJ71N10.1, Tyrosine-protein kinase receptor ETK1, 83, 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4-(4-dihexadecylaminostyryl)N-methylpyridium iodide, Immune, Methodological Studies, DIANA, 38E.16, DIA2, PrecipitationsPXD054173falseLINE-1 ORF1p co-immunoprecipitations analyzed by DDA and two DIA methodsUsing an anti-ORF1 monoclonal (clone 4H1; Milipore), four co-immunoprecipitations to enrich for LINE-1 ORF1p and its interactors as described in Di Stefano, L.H. et al. (https://doi.org/10.1007/978-1-0716-2883-6_12), from either HEK-293TLD (cells transfected with pMT646- containing a L1 sequence) or N2102Ep (embryonal carcinoma cells with endogenous expression of LINE-1) lysates were then analyzed in DDA, HRMS1, and Variable Window modes. Mouse polyclonal IgG were used as mock IPs as nonspecific binding controls. MMTS alkylated the cysteines. DDA and Variable Window experiments collected spectra for 60 minutes of the one hour run time. The HRMS1 experiments collected spectra for 90 minutes of a two hour run time. The UP000005640 reference proteome for Homo sapiens, including unreviewed sequences and reviewed isoforms was used as a search space (104,558 sequences)Wed Jul 24 02:39:00 BST 2024MSV00009542739372605