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EggersMass SpectrometryShotgun proteomicsSWATH MSNot availableLaser microdissectionProteomicsNeuromuscular disordersFiber typesSkeletal musclehttp://www.ebi.ac.uk/pride/archive/projects/PXD025359Skeletal Muscle FiberSkeletal Muscle TissueMice were sacrificed and skeletal muscle specimens were collected and ifrozen in isopentane pre-cooled in liquid nitrogen. Soleus and tibialis anterior muscle were cryosected into 10 µm thick slices (Cyrostat Microm HM550, Thermofisher Scientific, Schwerte Germany) and were placed on a PET membrane frame slide for laser microdissection (LMD) and stained with antibodies directed against the specific MYH isoforms. Fiber types were excised seperately using laser microdissection (LMD; 6500, Leica Microsystems, Wetzlar, Germany), excising a total of 1,000,000 µm² (correspoding to approximately 500 excised fibers). Samples were subsequently lysed with 40 µL of formic acid and incubated for 20 min at RT. Sonication of samples for 5 min lead to tissue and PET membrane disruption. Afterwards, formic acid was completely removed by vacuum vaporization (Vacuum concentrator RCV2-25 CD Plus, Martin Christ Gefriertrocknungsanlagen, Osterode, Germany) and the total digestion volume was set to 50 µL using 50 mM Ammonium-Bicarbonate and 5 mM 1,4-Dithiothreitol (DTT) (end concentration). Reduction with DTT was performed for 20 min at 56°C followed by alkylation for 15 min in the dark at room temperature with 15 mM IAA (end concentration). Tryptic digestion was carried out overnight, using 0.1 µg of trypsin per sample, and stopped after 16 h by adding 0.5 % trifluoroacetic acid (TFA) (end concentration). Sample volume was completely vaporized in a vacuum concentrator and peptides were solved in 50 µL 0.1%. Whole muscle tissue was prepared for spectral library generation. Here, complete murine soleus and tibialis anterior muscles were pulverized in liquid nitrogen, homogenized on ice, and afterwards resuspended in urea buffer (7M urea, 2M thiourea, 20mM Trisbase, pH 8,5). Resuspended samples were sonicated six times for ten seconds, with ten seconds rest on ice to support the lysis. Protein concentration was determined by Bradford assay. Nano HPLC analysis was performed on an UltiMate 3000 RSLC nano LC system (Thermofisher Scientific, Bremen, Germany). The HPLC system was online-coupled to the nano ESI source of a QExactive HF mass spectrometer (Thermofisher Scientific, Bremen, Germany). Prior MS analysis labeling of samples by internal retention time (iRT) calibration was performed for the evaluation of data with SpectronautTM Pulsar (Biognosys, Schlieren Switzerland). iRT peptides were prepared as recommended by the vendor. For DDA analysis, full MS spectra were acquired in a range from 350 to 1,400 m/z with a resolution of 60,000 at 200 m/z for the detection of precursor ions (AGC target 3e6, 80 ms maximum injection time). The m/z values initiating MS/MS were set on a dynamic exclusion list for 30 s, and the ten most intensive ions (charge state +2, +3, +4) were selected for fragmentation. MS/MS fragments were generated by high-energy collision-induced dissociation (HCD). Normalized collision energy (NCE) was either set to a fixed value of 27 or a stepped NCE was applied (25.5, 27, 30). The fragments were analysed in an Orbitrap analyser with 30,000 resolution at 200 m/z (AGC 1e6, maximum injection time 120 ms). In the ESI-MS/MS analysis, full MS spectra were scanned in a range between 350 and 1,400 m/z with a resolution of 120,000 at 200 m/z for the detection of precursor ions (AGC target 3e6, 20 ms maximum injection time). MS/MS fragments were generated by high-energy collision-induced dissociation (HCD) in which ion dissociation was performed at a NCE of 27%, fixed first mass of 130.0 m/z and 24 isolation windows of 45 m/z. The fragments were analysed in an Orbitrap analyser with 30,000 resolution at 200 m/z (AGC 1e6, maximum injection time 120 ms). For DIA analysis 8 µL sample volume were used. In the ESI-MS/MS analysis, full MS spectra were scanned in a range between 350 and 1,400 m/z with a resolution of 120,000 at 200 m/z for the detection of precursor ions (AGC target 3e6, 20 ms maximum injection time). MS/MS fragments were generated by high-energy collision-induced dissociation (HCD) in which ion dissociation was performed at a NCE of 27%, fixed first mass of 130.0 m/z and 24 isolation windows of 45 m/z. The fragments were analysed in an Orbitrap analyser with 30,000 resolution at 200 m/z (AGC 1e6, maximum injection time 120 ms).PrideData-independent acquisitioniodoacetamide derivatized residuemonohydroxylated residuedeamidated residueSpectral library generation was carried out using SpectronautTM Pulsar and the Pulsar search engine (v. 12.0.20491.7.17149, Biognosys, Schlieren, Switzerland). Data were searched against the Uniprot KB mus musculus reference proteome set including iRT peptides (version 2018_06, 53,560 entries) and a contaminant database resulting in a library size of 28,004 peptides and 5,206 proteins. Biognosys factory settings were applied and trypsin was chosen as digestion enzyme. Specific modifications were set according to sample treatment: carbamidomethylation (C) (fixed modification) and oxidation of (M), deamidation (NQ) and carbamidomethylation (N-term) (variable modifications) were included. After exporting the unnormalized protein and peptide intensities out of Spectronaut Pulsar, a locally weighted scatter plot smoothing normalization (LOESS) was applied using the limma R package version 3.36.5. Calculation of protein quantities was done using the aLFQ package (version 1.3.5) within R (version 3.6.1). First, for each protein an iBAQ value was calculated from the LOESS-normalized peptide intensities. Absolute protein quantities were obtained using the known total protein concentration of 200 ng per sample. In aLFQ, this is achieved by dividing all iBAQ values over all samples by the total sum of iBAQ values and multiplying with the specified total protein concentration. We modified the aLFQ workflow and conducted the protein quantification step separately for each sample, to ensure a total normalized protein intensity of 200 ng in each sample.ProteomicsKatrin MarcusQ Exactive HFMedizinisches Proteom-Center, Medical Faculty, Ruhr-University Bochum, 44801 Bochum, Germany Medical Proteome Analysis, Center for Proteindiagnostics (PRODI), Ruhr-University Bochum, 44801 Bochum, GermanyPARTIALMus Musculus (mouse)britta.eggers@rub.deNot availableRuhr University BochumGermanysodium salt, CPD photolyase activity, CDF, ammonium formate, 13C-labeled, cadmium salt, IL1BC, PhrB photolyase activity, ion, Laboratory, TFA, CASP-1, Longterm., magnesium formate, 2-(indol-3-yl)ethanoic acid, Basodexan, Apaf-1, House Mouse, zinc salt, Myh, MYH, Long Term, 5730420M11Rik, Trifluoroacetate, Polypeptides, cobalt(II) formate dihydrate, azanium, Microdissections, Urea, 3, AGAMOUS-like 61, NUP96, outer pigmented layer of retina, Il1bc, epithelium, WMS, myh, DiA, SET, hac-1, N, fibula, phosphatase 2A inhibitor I2PP2A, Tissue, DIASP, IL-1BC, DIA, Dia, HPLC, SUPPRESSOR OF AUXIN RESISTANCE 3, AI047805, DmelCG4299, isolation and purification, 4-(4-dihexadecylaminostyryl)N-methylpyridium iodide, set, pigment epithelium of retina, scientific observation, sample, n, pigmented retina epithelium, autolysin activity, z, Ammonium(1+), nickel salt, SGS, Caspase-1 subunit p10, cobalt (+2) salt, anon-53Fa, Longterm Effect, integral to membrane, l(2)SH0173, set of skeletal muscles, antibodies, not genetically inherited, collisionally activated dissociation, CID, DmelCG7788, ACMICD, (Indol-3-yl)acetate, sodium (4:1:1) salt, Stickstoff, magnesium salt, Dm1, dipyrimidine photolyase (photosensitive), simple tissue, END, CG6829, soleus, dark/hac-1/dapaf-1, Acid, HLA-DR-associated protein II, Caspase-14 subunit p10, DI-2, soleus muscle, I-2Dm, F27C12_24, mesomelic dwarfism, Swiss Mouse, formate, F27C12.24, cromium (+3), Caspase-14 subunit p19, inhibitor of granzyme A-activated DNase, beta Trypsin, I-2PP1, Indole-3-acetic acid, nitrogen, TAF-IBETA, heteroauxin, 2-methylbutane, Langer type, Drice, DIANA, ammonium (4:1) salt, bacteriolytic toxin activity, Mouse, TAF-Ibeta, lead (+2) salt, DRICE, tibialis anterior muscle, AA407358, 3.4.22.-, nickel formate dihydrate, lead formate, Effects, DrIce, DrICE, aluminum salt, muscles, mini-ICE, Carbamide, precursor, thomson, body system, deoxyribodipyrimidine photolyase activity, pet, CG7826, stratum pigmentosum retinae, tibilais cranialis, mass-to-charge ratio, period, ammonium cation, isolation, CG7835, HILDA, Mus musculus domesticus, CG42273, system, Carmol, Cesium Trifluoroacetate, fibula and mandible type, anatomical systems, thallium (+1) salt, Longterm, Tissues, rubidium salt, beta-Trypsin, PCE-2, F23A5.3, 2pp2a, Long-Term, methanoic acid, ESI, ions, study assay, CG10574, Injectable, 2PP2A, laser-assisted microdissection, Caspase-1 subunit p20, dSET, dSet, ibialis anticus, peptidos, striated muscle, hyt, 1H-indol-3-ylacetic acid, measuring, dark/dapaf-1/hac-1, tibialis cranialis, grupo, caspase 3, pseudoautosomal recessive, dApaf-1, NH4+, potassium formate, copper (+2) salt, CE-2, DRF2, buffer, CD142, polypeptide, APAF1, Temperatures, I-2PP2A, Hac-1, carbonyldiamide, cupric formate, Dm I-2, chemical analysis, Long Term Effects, CYP2C, MFS1, LCM, aluminum formate, Mutyhalpha, Library, AU020952, muscular tissue, RPE, lithium formate, muscles set, holin, 3-Indolylessigsaeure, Dark/Dapaf-1/HAC1, House Mice, IL-1 beta-converting enzyme, azote, Interleukin-1 beta-converting enzyme, retinal pigment epithelium, Longterm Effects, p. pigmentosa retinae, ME-IV, deoxyribonucleate pyrimidine dimer lyase (photosensitive), and mandible type, langer mesomelic dysplasia, Muscle Tissue, Hac-1/Dark, Mutyhc, IPP2A2, muscle system, Mutyha, determination, Mutyhb, DmelCG6829, Mus domesticus, zinc formate, CG7788, crice, lead salt, CASP-14, pigmented epithelium, lysis, Alkylations, peptide, portion of muscle tissue, ammonium tetraformate, POF, IAA, peptido, Min, Apaf1, ARK, Injectables, Effect, DmelCG42273, portion of tissue, Muscle Tissues, l(2)k07135, peptides, TAF-I, Rests, E927b, membrane region, homozygous, pigmented retina, min, arc, Swiss Mice, mAPC, purification, copper, ark, DNA cyclobutane dipyrimidine photolyase activity, T1, IGAAD, dyschondrosteosis, lithium salt, DmelCG10574, ICE, trifluoro-, 38E.16, TF, dApaf1, necrosis, GPHYSD2, ammonium (2:1) salt, Long-Term Effects, Langer mesomelic dysplasia, phapii, PRE, D-Apaf-1, ice, iCE, drice, set of muscles, deoxyribonucleic cyclobutane dipyrimidine photolyase activity, mouse, Dmel_CG7826, StF-IT-1, Th, pigmented retinal epithelium, membranous organ component, ur, Langer syndrome, any method, NH4(+), carbamide, ms(2)04138, Desmoplastic infantile astrocytoma, HCD, retinal pigment, XBR, Ionen, AI481368, drIce, drICE, Mini-ICE, tissue portion, bacteriocin activity, IES, nickel (+2) salt, fixed, retinal pigment layer, Dmel_CG7835, Acetic acid, Mnb, MNB, parent ion, Mus musculus, ammonium salt, membrane of organ, mice, retinal pigmented epithelium, Sonications, POF2, P45, Trifluoroacetic, cobaltous formate, CG4299, hac1, MASS, AW124434, MICE, domesticus, organ system, dapaf-1S, LMD, phr A photolyase activity, H2NC(O)NH2, apaf-1, dapaf-1L, lysin activity, DNA-photoreactivating enzyme, precursor ion, Cesium, copper salt, p45, Polypeptide, 7N, i2pp2a, 1728, time, Gruppe, Ammonium, Dapaf-1/HAC-1, cesium salt, musculature system, FBN, Mutyhbeta, photoreactivating enzyme activity, skeletal muscle, [NH4](+), PHAPII, formic acid, Buffer, Dark/Hac-1/dApaf1, Karbamid, Hac1, potassium salt, Dark/Hac-1/dApaf-1, template-activating factor I, House, Injection, ECTOL1, integral component of membrane, 14C-labeled, DmelCG1768, Harnstoff, NRSF, stratum pigmentosa retinae, Mice, hypothroid, MOS3, anterior tibialis, deoxyribocyclobutadipyrimidine pyrimidine-lyase activity, PRECOCIOUS, Swiss, iones, ammonium ion, ipp2a2, dapaf-1, dapaf, Indoleacetic acid, labeling, dark, calcium formate, OCTD, (indol-3-yl)acetic acid, DARK, taf-ibeta, grupos, region of membrane, liquid, cromium (+3) salt, Long-Term Effect, DIA2, dArk, Dapaf-1, textus muscularis, F23A5_3, sodium formate, HHT1, musculus tibialis anterior, membrane, DYRK1, data, nickel formate, musculi, Desmoplastic astrocytoma of infancy, igaad, 2610008J04Rik, strontium formate, TelN, apaf1, CG1768, 5730495A01Rik, strontium salt, Muscle, Peptide, light amplification by the stimulated emission of radiation, group, somatic muscle, stratum pigmentosum (retina), MODIFIER OF SNC1, CAD, Mus, MLPLI, deoxyribonucleic photolyase activity, I2PP2A, whole membrane, Dyrk1, Vacuums, Dark, CES2A1, connected anatomical system, Dias, Dark/Apaf-I, WMS2, transmembrane, 10^[-9], ammonium, photolyase activity, uree, urea, CC1, Tripcellim, Rest, Langer type mesomelic dysplasia, dApaf-1/DARK/HAC-1, chromic formate, sample population, UREA, Laboratory Mice, nitrogeno, Interleukin-1 beta convertase, Trypure, dSET/TAF-Ibeta, 2610030F17Rik, Ion, concentration, SSKS, 3.4.22.36, Calibrations, calcium salt, mesomelic dwarfism of the hypoplastic ulna, musculature, ResT, Peptid, assay, ORW1, AA407739, Laboratory Mouse, groupe, dAPAF-1, muscle group, skeletal muscle systemIPP2A2, GRP1/cytohesin 1, scattergraph, mouse <Mus musculus>, Gene, protein, protein-containing complex, PHAPII, ter, 5730420M11Rik, CG11633, peptide, cytohesin/GRP1, Polypeptides, protein polypeptide chains, peptido, ClvPrd, template-activating factor I, polypeptide chain, GRP1, Grp1, Gene Products, protein aggregate, Work Flow, Search Engines, SET, peptides, TAF-I, phosphatase 2A inhibitor I2PP2A, PTPSTEP, ipp2a2, beta-Trypsin, l(2)SH2 0323, 2pp2a, proteins, CG10574, DmelCG4299, set, IGAAD, DmelCG4216, 2PP2A, Neural-specific protein-tyrosine phosphatase, DmelCG10574, taf-ibeta, Bsg75C, l(2)k08110, sample, dSET, dSet, peptidos, house mouse, GPH, phapii, data, protein complex, Proteins, mouse, igaad, total expressed protein, StF-IT-1, stepk, Search, Peptide, Engine, polypeptide, Workflows, native protein, natural protein, I-2PP2A, Term, Dm I-2, Protein, I2PP2A, 3.1.3.48, l(2)SH0323, Library, Data Base, CYH1, HLA-DR-associated protein II, DI-2, Step, CG11628, I-2Dm, Tripcellim, CG4216, Striatum-enriched protein-tyrosine phosphatase, CG4299, inhibitor of granzyme A-activated DNase, beta Trypsin, sample population, DmelCG11628, I-2PP1, Protein Gene Products, Gene Proteins, Trypure, dSET/TAF-Ibeta, sample., 2610030F17Rik, TAF-IBETA, concentration, STEP, 2018, Peptid, TAF-Ibeta, Polypeptide, variable, AA407739, i2pp2a, Proteomes, Work FlowsMPC, other disease, advanced, portion of heterogeneous tissue, muscle system, Activity, determination, CG 1618, Blood, Training, musculature system, Isometric, Physical, muscles, A4, FBN, Comt, dNSF, Gene, NSF1, Exercise Training, protein, hemangiopericytoma, Spectrum Analyses, protein-containing complex, composed of, skeletal muscle, TYPE, NSF, Nsf, DAGA4, peptide, Polypeptides, method, protein polypeptide chains, CG1618, blood vessels, DmNSF, ClvPrd, peptido, diseases, polypeptide chain, Physical Exercises, ECTOL1, portion of connective tissue, method used in an experiment, Mass, Gene Products, disease or disorder, dNSF1, muscular disease, dNsf1, diseases and disorders, Analysis, MAM, protein aggregate, Connective Tissues, Acute Exercises, SCG3, WMS, Mass Spectroscopy, Phenomenography, Mass Spectrum Analysis, textus connectivus, Aerobic Exercises, human disease, Muscle Tissues, peptides, Analyses, Tissues, Physical Activity, Tissue, Blood Vessel, composition, proteins, Connective, free, non-neoplastic, OCTD, musculus, DmelCG1618, disorder, Homo sapiens disease, peptidos, associated, GPHYSD2, Exercises, SGS, striated muscle, com, Data Set., blood vessel system, musculi, set of muscles, protein complex, Proteins, disorders, Exercise, Aerobic, set of skeletal muscles, medical condition, function, compositionality, Muscle, Spectrum Analysis, blood vascular network, Peptide, light amplification by the stimulated emission of radiation, LGMD2C, ACMICD, Spectroscopy, polypeptide, somatic muscle, Muscle Disorders, native protein, natural protein, chemical analysis, Protein, Diseases, precocious, condition, MFS1, Acute Exercise, Isometric Exercises, dNSF-1, Physical Exercise, Mass Spectrum Analyses, WMS2, dnsf1, Aerobic Exercise, Mass Spectrum, Bindegewebe, muscles set, solitary myofibroma, DMDA1, content, blood system, Vessel, Spectrometry, muscle, MASS, Trainings, study protocol, muscle element, Exercise Trainings, early, Isometric Exercise, Activities, plan specification, Protein Gene Products, Gene Proteins, disease, myopathy, Vessels, Acute, DMDA, Physical Activities, set of blood vessels, structure, SCARMD2, SSKS, musculature, Peptid, NSF-1, assay, Polypeptide, Muscle Tissue, hypothesis, muscle group, skeletal muscle system, Myopathyskeletal muscle system., advanced, adults, DMDA1, protein complex, adult stage, A4, proteins, protein, protein-containing complex, skeletal muscle, TYPE, early, LGMD2C, DAGA4, protein polypeptide chains, somatic muscle, DMDA, native protein, natural protein, polypeptide chain, SCARMD2, Protein, precocious, MAM, protein aggregate, SCG3, Adults, adult, striated musclefalseAdvanced fiber type-specific protein profiles derived from adult murine skeletal muscleSkeletal muscle is a heterogeneous tissue consisting of blood vessels, connective tissue, and muscle fibers. The last are highly adaptive and can change their molecular composition depending on external and internal factors, such as exercise, age, and disease. Thus, examination of the skeletal muscles at the fiber type level is essential to detect potential alterations. Therefore, we established a protocol in which myosin heavy chain isoform immunolabeled muscle fibers were laser microdissected and separately investigated by mass spectrometry to develop advanced proteomic profiles of all murine skeletal muscle fiber types. Our in-depth mass spectrometric analysis revealed unique fiber type protein profiles, confirming fiber type-specific metabolic properties and revealing a more versatile function of type IIx fibers. Furthermore, we found that multiple myopathy-associated proteins were enriched in type I and IIa fibers. To further optimize the assignment of fiber types based on the protein profile, we developed a hypothesis-free machine-learning approach (available at: https://github.com/mpc-bioinformatics/FiPSPi), identified a discriminative peptide panel, and confirmed our panel using a public data 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