MassIVEapplication/xmlftp://massive-ftp.ucsd.edu/v04/MSV000088805/primaryOK2000000Mark A. McGuirehttps://massive.ucsd.edu/ProteoSAFe/dataset.jsp?task=8cfdbc31839440bd82ace7d13a2c20f2mmcguire@uidaho.eduMassIVE73UNIMOD:21 - "Phosphorylation."UNIMOD:1 - "Acetylation."UNIMOD:35 - "Oxidation or Hydroxylation."ProteomicsOrbitrap EclipseOrbitrap Fusion LumosQ Exactive HFBos Taurus (ncbitaxon:9913)University of IdahoGenerating top-down tandem mass spectra (MS/MS) from complex mixtures of proteoforms benefits from improvements in fractionation, separation, fragmentation, and mass analysis. The algorithms to match MS/MS to sequences have undergone a parallel evolution, with both spectral alignment and match-counting approaches producing high-quality proteoform-spectrum matches (PrSMs). This study assesses state-of-the-art algorithms for top-down identification (ProSight PD, TopPIC, MSPathFinderT, and pTop) in their yield of PrSMs while controlling false discovery rate. We evaluated deconvolution engines (ThermoFisher Xtract, Bruker AutoMSn, Matrix Science Mascot Distiller, TopFD, and FLASHDeconv) in both ThermoFisher Orbitrap-class and Bruker maXis Q-TOF data (PXD033208) to produce consistent precursor charges and mass determinations. Finally, we sought post-translational modifications (PTMs) in proteoforms from bovine milk (PXD031744) and human ovarian tissue. Contemporary identification workflows produce excellent PrSM yields, although approximately half of all identified proteoforms from these four pipelines were specific to only one workflow. Deconvolution algorithms disagree on precursor masses and charges, contributing to identification variability. Detection of PTMs is inconsistent among algorithms. In bovine milk, 18% of PrSMs produced by pTop and TopMG were singly phosphorylated, but this percentage fell to 1% for one algorithm. Applying multiple search engines produces more comprehensive assessments of experiments. Top-down algorithms would benefit from greater interoperability.Comparing Top-Down Proteoform Identification: Deconvolution, PrSM Overlap, and PTM Detection.Tabb David L DL, Jeong Kyowon K, Druart Karen K, Gant Megan S MS, Brown Kyle A KA, Nicora Carrie C, Zhou Mowei M, Couvillion Sneha S, Nakayasu Ernesto E, Williams Janet E JE, Peterson Haley K HK, McGuire Michelle K MK, McGuire Mark A MA, Metz Thomas O TO, Chamot-Rooke Julia JTaurine, dairy cow, Inflammatory disorder of breast (disorder), Beef Cows, multi-cellular organism, cow, Taurus Cattles, Bos indicus Cattles, body, Holstein Cow, animalia, inflammation of breast, Taurus Cattle, total expressed protein, Cattle, Taurine Cattles, whole body, Indicine, Bos indicus, Dairy Cows, Inflammatory disease of breast, Milk, mammary gland milk, organism, Bos grunniens, Tier, inflammatory disease of breast, cattle, Bos taurus, Bos primigenius taurus, Cow's Milk, Metazoa, Cow's, breast inflammation NOS, domestic cattle, Indicine Cattles, Animal, Yaks, animal, Holstein, Cattles, Domestic Cows, Animalia, Yak, Cows, whole organism, Inflammatory breast disease, Domestic Cow, inflammatory breast disease, Dairy Cow, Bos bovis, metazoa, Domestic, Dairy, Taurine Cattle, Beef, Mastitis, Bos Tauurus, mastitis, Beef Cow, oxen, breast inflammation, ox, Cow, Cow Milk, Taurus, Koerper, Indicine Cattle, breast infection., breast inflammation NOS (disorder), Zebu, domestic cow, Zebus, Bos indicus Cattle, bovine, Proteomessodium salt, Water, dairy cow, Inflammatory disorder of breast (disorder), ammonium formate, 13C-labeled, cadmium salt, Molecular Weights, Laboratory, H(2)O, BOUND WATER, magnesium formate, oxidane, Inflammatory disease of breast, zinc salt, heavy chain disease, Solvent, Milk, breast infection, WATER, clot, HOH, 5730420M11Rik, cobalt(II) formate dihydrate, Method, Cow's, WMS, Animalia, average, SET, Divorced, C79325, Cows, Molecular, Inflammatory breast disease, Domestic Cow, Dairy Cow, Tissue, Weights, HardwareType, Divorces, procedures, T6G21.3, Beef, AI047805, DmelCG4299, Bos Tauurus, set, oxen, breast inflammation, column, ACN, blood, Acn, sample, nickel salt, SGS, NK/GPI, Gpi, Stars, cobalt (+2) salt, Taurus Cattles, animalia, integral to membrane, Normalcy, Procedure, Bos indicus, ACMICD, H2O, sodium (4:1:1) salt, Dm1, magnesium salt, Weight, NK|GPI, Holstein, Hydrogen Oxide, whole organism, HLA-DR-associated protein II, instrument, DI-2, I-2Dm, formate, cromium (+3), Methodological, study protocol, I-2PP1, Grant, Mastitis, mastitis, TAF-IBETA, Charges and Fees, Koerper, ammonium (4:1) salt, Zebu, TAF-Ibeta, Bos indicus Cattle, bovine, lead (+2) salt, STARS, Bglap-rs1, Community, multi-cellular organism, nickel formate dihydrate, lead formate, Peptidomics, aluminum salt, number, precursor, Doe, Normalcies, 17alpha)-isomer, CG7826, DOI, agua, Cow's Milk, Gene Products, CG7835, CG42273, Pgi, Indicine Cattles, Financing, Technique, doe, doi, Normalities, Community Financing, Acinus, thallium (+1) salt, Gpi-1r, Tissues, Gpi-1s, Postpartum, Phi, Side, rubidium salt, Gpi-1t, 2pp2a, Sciences, uL/min, Dairy, methanoic acid, Taurine Cattle, CG12510, CG10574, CD156, acinusL, Study, 2PP2A, acinusS, Cow Milk, Cow, Grants, dSET, dSet, ETD, Individual Health, cow, Bos indicus Cattles, body, Holstein Cow, Proteins, Taurine Cattles, whole body, potassium formate, copper (+2) salt, MF, backward, Amf, mKIAA0670, CG13979, mammary gland milk, CG13974, Bos grunniens, CG13975, CG31062, inflammatory disease of breast, Experiment, I-2PP2A, Bos primigenius taurus, cupric formate, chemical analysis, Dm I-2, 5alpha, MFS1, domestic cattle, aluminum formate, CG13973, Cattles, NK, AU020952, lithium formate, United States of America, ensemble, Separations, Striated muscle activator of Rho-dependent signaling, CD156a, cardinality., ME-IV, plan specification, Gene Proteins, Clots, ox, Fees, breast inflammation NOS (disorder), [OH2], reversed, Taurine, IPP2A2, determination, inflammation of breast, Taurus Cattle, instrument configuration, zinc formate, lead salt, Dairy Cows, Thromboses, Membrane Tissues, Techniques, Tier, ammonium tetraformate, BANF, Min, Gpi-1, animal, DmelCG42273, Yak, 3-hydroxy-5-estrane-17-carbonitrile, TAF-I, thrombus, Nlk, inflammatory breast disease, 2610036I19Rik, membrane region, Bos bovis, 2610510L13Rik, E430039A18Rik, min, U.S., mAPC, Domestic, copper, microlitres per minute, IGAAD, fSAP152, lithium salt, Methodological Studies, DmelCG10574, dihydridooxygen, domestic cow, Zebus, dihydrogen oxide, GPHYSD2, ammonium (2:1) salt, blood clot, phapii, Membrane Tissue, Beef Cows, DmelCG31062, aqua, Dmel_CG7826, StF-IT-1, Cattle, Indicine, membranous organ component, Programs, organism, mOC-X, HCD, Bos taurus, laboratory, Individual, Yaks, nickel (+2) salt, Dmel_CG7835, Mnb, MNB, parent ion, ammonium salt, Org, ORG, membrane of organ, CHARGES, MS/MS, cobaltous formate, hydrogen hydroxide, thrombosis, Gpi1-r, Gpi1-s, MASS, CG4299, AW124434, Gpi1-t, Methodological Study, acqua, Health, precipited material, precursor ion, Charge, Taurus, copper salt, Wasser, i2pp2a, Gpi1s, Procedures, AI461847, cesium salt, FBN, Gene, ACINUS, blood Clots, presence, PHAPII, hardware, Women, formic acid, method, potassium salt, cattle, Ms1, ECTOL1, integral component of membrane, 14C-labeled, method used in an experiment, Studies, Animal, Separated, Contract, Domestic Cows, MS1, MS2, Postpartum Women, ipp2a2, metazoa, eau, calcium formate, Charges, OCTD, Beef Cow, Fee, Suspension, taf-ibeta, male sterility 1, region of membrane, cromium (+3) salt, CT33528, CT33527, sodium formate, membrane, DYRK1, nickel formate, igaad, strontium formate, strontium salt, group, count in organism, Period, Organized Financing, water, Metazoa, Protein, I2PP2A, whole membrane, breast inflammation NOS, Dyrk1, techniques, (3alpha, tandem MS, WMS2, Separation, transmembrane, thrombotic disorder, MALE STERILITY 1 PROTEIN, Normality, CC1, Membrane, chromic formate, sample population, Protein Gene Products, dSET/TAF-Ibeta, 2610030F17Rik, Puerperium, SSKS, calcium salt, Indicine Cattle, assay, SCH, AA407739, methodology, 3-OHECNposttranslational amino acid modification, posttranslational modification, posttranslational protein modification, PTM, post-translational amino acid modification., post-translational modificationPost Translational Amino Acid Modification, dairy cow, human being, STAB-2, Post-Translational Protein Modifications, Extract, determination, FEZL, FBN, 9930121L06Rik, precursor, CG3629, Protein Processing, Crude Extracts, Milk, Posttranslational, Human, Crude, Crude Extract, Complex Mixture, l(2)387, Homo sapiens, TOF, cattle, ECTOL1, Cow's Milk, Post Translational Modification, Extracts, Cow's, Post-Translational Modifications, Work Flow, Separated, Man, WMS, ZNF312, Post Translational, ASD2, AI661365, Modification, Post-Translational Protein, study, Art, ART, Search Engines, Mixtures, Divorced, Man (Taxonomy), reference sample, Post-Translational, TACHD, MS2, Complex, Bos bovis, Tissue, TINT1, Sciences, Divorces, Charges, Posttranslational Protein Processing, Ba, FELL, OCTD, Protein Modifications, Post-Translational Amino Acid Modification, Bos Tauurus, oxen, Fee, Posttranslational Amino Acid Modification, Cow Milk, Cow, Posttranslational Protein, domestic cow, 2610009E16Rik, GPHYSD2, TPP1, FEZ, 2.7, SGS, Controlled, Post-Translational Modification, Controlling, dl, cow, Mixture, Arts, Modern, Modifications, Post-Translational Protein Modification, Search, Engine, ACMICD, l(2)01092, mammary gland milk, Workflows, Post Translational Modifications, Bos primigenius taurus, FEEL-2, DmelCG3629, Algorithm, chemical analysis, BcDNA:LP01770, MFS1, domestic cattle, simple tissue, FEEL2, IVF, Protein Modification, tandem MS, WMS2, PIP1, PTOP, parent ion, ParaT, VSD1, Separation, DLL, CHARGES, MFEEL-2, Algorithm., MS/MS, Post Translational Protein Processing, HARE, Separations, Processing, MASS, E(Arp), En(Arp), class, human, Complex Extract, ZFP312, dll, Post Translational Protein Modification, Snm1l, socioeconomic factors, precursor ion, Charge, Amino Acid Modification, ox, Fees, Charges and Fees, Modern Man, SSKS, Post-Translational Protein Processing, Posttranslational Modifications, assay, AGRT, bovine, FEX2, Posttranslational Modification, Work Flows, humans, Complex Extracts0PXD031744falseTop-down proteome of de-fatted Bos taurus milk from an animal with clinical mastitisQuarter-milk samples were collected from a cow with clinical mastitis on day 13 and day 16 postpartum. Milk was collected from a clinical quarter with signs of abnormality (i.e., flakes, clots, blood) and one normal quarter of the same animal. Milk samples were processed with the MPLEx protocol (http://dx.doi.org/10.1039/C6AN02486F) to precipitate the proteins. The pellet was re-suspended in 500uL 3% ACN, 0.1% formic and the suspension was then either filtered with Amicon 100k Da molecular weight cutoff membrane filter, or ultracentrifuged at 100 kg for 10 min. The filtrate or the supernatant, respectively, were diluted at 0.2 ug/uL in 3% ACN, 0.1% formic acid for LCMS analysis. A Waters NanoAcquity LC was used for online separation. The binary mobile phase solvents are 0.2% formic acid in water (A), and 0.2% formic acid in ACN (B). For each LCMS experiment, 5 uL sample was injected into the trapping column (in-house packed, 5 cm, inner diameter 150 um, outer diameter 360 um, C2 reversed phase particles, MEB2-3-300, Separation Methods Technologies). After washing with 5% mobile phase B at 3 uL/min for 5 min, sample was loaded on to the C2 analytical column (in-house packed, 50 cm, inner diameter 100 um, outer diameter 360 um, same packing material as the trap column) and separated with 5-50% mobile phase B gradient at 0.3 uL/min over 100 min. Top-down MS data were collected on Orbitrap based mass spectrometers with standard data dependent acquisition. The membrane filtered samples were analyzed on a Thermo Orbitrap Eclipse with higher-energy collision dissociation for MS2. The ultracentrifuged samples were analyzed on both a Thermo Q Exactive HF (HCD for MS2) and a Thermo Orbitrap Fusion Lumos (HCD and also ETD for MS2). The main data included here are both high resolution MS1 (120k) and high resolution MS2 (60k). Lower resolution MS1 data were collected for Q Exactive HF (15k) and Lumos (7.5k) instruments. These RAWs are included but not analyzed for this deposit. The average of MS/MS counts for the Orbitrap Eclipse experiments was 22,671, greatly outnumbering those of the Orbitrap Fusion Lumos (13,408) and of the Q-Exactive HF (8,820) for the same LC duration (the numbers of spectra reflect both sample processing and instrument method differences). The Eclipse set produced an interquartile range of +6 to +13 for its precursor charges, and the Fusion Lumos reported +6 to +12. The Q-Exactive HF, however, yielded a lower interquartile range of +5 to +9. Proteomics analyses were performed in the Environmental Molecular Sciences Laboratory, a national scientific user facility sponsored by the U.S. OBER and located at PNNL in Richland, Washington. PNNL is a multi-program national laboratory operated by Battelle for the DOE under Contract DE-AC05-76RLO 1830. Funding was provided by the National Institutes of Health grant number 1R01HD092297-01A1. Wed Feb 09 00:46:00 GMT 2022MSV00008880537235544