Layout 1 Thematic Section: Advances in Musculoskeletal and Neuromuscular Rehabilitation | Maccarone & Masiero Eur J Transl Myol 34 (2) 12565, 2024 doi: 10.4081/ejtm.2024.12565 Mass spectrometry (MS) based proteomics is a key method of modern protein chemistry,1 including skeletal muscle biochemistry,2 that can be employed for both the targeted or untargeted detection and characteriza- tion of specific proteins.3,4 The various molecular forms of a muscle protein product that is encoded by a particular gene and influenced by alternative RNA splicing of its tran- script,5 plus dynamic post-translational modifications,6 are referred to as proteoforms,7 which form the basic units of the proteome.8 Mass spectrometry can be used for compar- ative studies,9 or the systematic cataloguing of tissue-spe- cific protein constellations,10 such as the human skeletal muscle proteome.11-13 In principle, the main proteomic ap- proaches can be divided into bottom-up versus top-down analyses,14 which differ in their starting material prior to mass spectrometric analysis.15 Bottom-up proteomics is a gel-free and peptide-focused methodology for protein iden- tification,16,17 while top-down proteomics specializes on proteoform-centric and often the gel-based detection and characterization of intact protein species.18,19 To date, a variety of proteomic studies have focused on the characterization of skeletal muscle tissues using MS-based proteomics.12 This has included skeletal muscle specimens and its secretome from various species using both top-down and bottom-up proteomics.9,11,20-23 Systematic protein cata- loguing studies,24-27 have resulted in the identification of a large number of dynamic proteoforms that are characteristic for voluntary striated muscles.11 The proteomic screening of contractile tissues has revealed crucial information on myofiber types in slow versus fast muscles,28-31 the trans- formation of muscles due to changed activity patterns,32-34 the impact of physical activity,35-38 the pathophysiological Abstract During the natural aging process, frailty is often associated with abnormal muscular performance. Although inter-individual differences exit, in most elderly the tissue mass and physiological functionality of voluntary muscles drastically decreases. In order to study age-related contractile decline, animal model research is of central importance in the field of biogerontology. Here we have analyzed wild type mouse muscle to establish a proteomic map of crude tissue extracts. Proteomics is an advanced and large-scale biochemical method that attempts to identify all accessible proteins in a given biological sample. It is a technology-driven approach that uses mass spectrometry for the characterization of individual protein species. Total protein extracts were used in this study in order to minimize the potential introduction of artefacts due to excess subcellular fractionation procedures. In this report, the proteomic survey of aged muscles has focused on organellar marker proteins, as well as proteins that are involved in cellular signaling, the regulation of ion homeostasis, bioenergetic metabolism and molecular chaperoning. Hence, this study has establish a proteomic reference map of a highly suitable model system for future aging research. Key Words: aging, mass spectrometry, muscle proteomics, skeletal muscle. Eur J Transl Myol 34 (2) 12565, 2024 doi: 10.4081/ejtm.2024.12565 Proteomic reference map for sarcopenia research: mass spectrometric identification of key muscle proteins of organelles, cellular signaling, bioenergetic metabolism and molecular chaperoning Paul Dowling,1,2 Stephen Gargan,1,2 Margit Zweyer,3,4 Michael Henry,5 Paula Meleady,5 Dieter Swandulla,6 Kay Ohlendieck1,2 1Department of Biology, Maynooth University, National University of Ireland, Maynooth, Co. Kildare, Ireland; 2Kathleen Lonsdale Institute for Human Health Research, Maynooth University, Maynooth, Co. Kildare, Ireland; 3Department of Neonatology and Paediatric Intensive Care, Children’s Hospital, University of Bonn, Bonn, Germany; 4German Center for Neurodegenerative Diseases, Bonn, Germany; 5National Institute for Cellular Biotechnology, Dublin City University, Dublin, Ireland; 6Institute of Physiology, Medical Faculty, University of Bonn, Bonn, Germany. This article is distributed under the terms of the Creative Commons Attribution Noncommercial License (CC BY-NC 4.0) which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. - 85 - Non -co mmerc ial us e o nly Proteomic reference map for sarcopenia research Part 2 Eur J Transl Myol 34 (2) 12565, 2024 doi: 10.4081/ejtm.2024.12565 effects of primary muscular disorders,39-41 dysfunctions of the musculature in the context of co-morbidities,42-44 and the natural aging process.45-50 The underlying objective of this study was to establish a proteomic reference map of a highly suitable model system for aging research of the neuromuscular system. Crude pro- tein extracts from normal tissue specimens of 100 mg wet tissue weight were used to establish a methodological ap- proach for the optimum utilization of small muscle biopsy specimens. In addition, a minimum of preparative steps was employed to simplify the overall experimental bottom-up protocol, accelerate and streamline the proteomic pipeline procedure and avoid the introduction of potential bioana- lytical artefacts for the routine detection of key protein species in aged skeletal muscles. This report has focused on the detailed proteomic analysis of aged skeletal muscles with specific emphasis on proteins that are intrinsically in- volved in cellular signaling mechanisms, the regulation of ion homeostasis, excitation-contraction coupling, bioener- getic metabolism and molecular chaperoning. In addition, proteomic markers of organelles, such as the sarcolemma, caveolae, transverse tubules, sarcoplasmic reticulum, triad junctions, ribosomes, Golgi apparatus, peroxisomes, pro- teasomes, lysosomes, nucleus, cytosol, the neuromuscular junction, motor neurons and glial cells, were investigated. Materials and Methods Materials Analytical grade chemicals from GE Healthcare (Little Chalfont, Buckinghamshire, UK), Sigma Chemical Com- pany (Dorset, UK) and Bio-Rad Laboratories (Hemel- Hempstead, Hertfordshire, UK) were used for the proteomic analysis of aged mouse hindlimb muscles. MS- grade trypsin, protease inhibitors (cOmplete™, mini pro- tease inhibitor cocktail) and spin filters (Vivacon 500, VN0H22; 30,000 MWCO) were from ThermoFisher Sci- entific (Dublin, Ireland), Roche (Mannheim, Germany) and Sartorius (Göttingen, Germany), respectively. The deter- mination of protein concentration was carried out with the Pierce 660 nm Protein Assay Reagent from ThermoFisher Scientific (Dublin, Ireland). Senescent murine hindlimb muscle Hindlimb muscles were dissected from freshly prepared post mortem specimens from 24 months old wild type C57BL6 mice according to institutional regulations. Mouse populations were handled in strict adherence to local gov- ernmental and institutional animal care regulations and were approved by the Institutional Animal Care and Use Committee (Amt für Umwelt, Verbraucherschutz und Lo- kale Agenda der Stadt Bonn, North Rhine-Westphalia, Ger- many).27 Mice were kept under specific pathogen-free conditions in isolated, ventilated cages with free access to water and food. A maximum of 4 mice were kept per cage at a temperature of 21-22oC with a 12-hour light/12-hour dark cycle. Tissue samples were quick-frozen in liquid ni- trogen and transported on dry ice to Maynooth University in accordance with the regulations of the Department of Ag- riculture (animal by-product register number 2016/16 to the Department of Biology, National University of Ireland, Maynooth).51 The mass spectrometric analysis of total skeletal muscle preparations was performed with protein extracts from 6 wild type mice. Protein extraction from muscle specimens Total protein extracts were used for the bottom-up pro- teomic analysis of aged hindlimb muscles from wild type C57BL6 mice.41 Standardized procedures were employed for optimum muscle tissue preparation, homogenization with help of pulverization of specimens by grinding in liq- uid nitrogen and subsequent protein extraction.52 Tissue ho- mogenization was carried out in lysis buffer, consisting of 0.1 M dithiothreitol, 4% (w/v) sodium dodecyl sulfate, 100 mM Tris-Cl, pH 7.6. The solution was supplemented with a protease inhibitor cocktail.41 The suspensions were briefly treated in a sonicating water bath and then heated for 3 min at 95oC.53 Samples were then centrifuged at 16,000×g for 5 minutes and the protein-containing supernatant extracted for mass spectrometric analysis. Protein determination was carried out with the Pierce 660 nm Protein Assay system.53 Protein samples were mixed with 8 M urea, 0.1 M Tris, pH 8.9 in Vivacon 500 spin filter units.54 Following a centrif- ugation step at 14,000×g for 15 min, the further processing of samples, the switching of buffers and peptide generation by trypsin digestion was carried out by the filter-aided sam- ple preparation (FASP) technique.55 Proteomic analysis For the label-free liquid chromatography mass spectromet- ric analysis of senescent hindlimb muscles from normal mice (n=6 biological repeats; n=2 technical repeats), a Thermo Orbitrap Fusion Tribrid mass spectrometer from Thermo Fisher Scientific (Waltham, MA, USA) was used.41 The detailed description of all analytical steps, including the listing of buffer composition, FASP protocol, timing of preparative stages, data-dependent acquisition, and bio- informatic data handling, used during the proteomic analy- sis of muscle proteins, has been described in a recently published methods paper.56 A Thermo UltiMate 3000 nano system was used for reversed-phase capillary high-pressure liquid chromatography and directly coupled in-line with the Thermo Orbitrap Fusion Tribrid mass spectrometer. The qualitative data analysis of mass spectrometric files was carried out with the UniProtKB-SwissProt database (species: Mus musculus) with Proteome Discoverer 2.2 using Sequest HT (Thermo Fisher Scientific) and Perco- lator. Parameters for the mass spectrometric detection of hindlimb muscle proteins were as follows: i) a value of 0.02 Da for MS/MS mass tolerance, ii) a value of 10 ppm for peptide mass tolerance, iii) variable modification settings for methionine oxidation, iv) fixed modification settings in relation to carbamido-methylation and v) tolerance for the occurrence of up to two missed cleavages. Peptide prob- ability was set to high confidence. A minimum XCorr score of 1.5 for 1, 2.0 for 2, 2.25 for 3 and 2.5 for 4 charge state was employed for the filtering of peptides. The software analysis programme Progenesis QI for Proteomics (version 2.0; Nonlinear Dynamics, a Waters company, Newcastle - 86 - Non -co mmerc ial us e o nly Proteomic reference map for sarcopenia research Part 2 Eur J Transl Myol 34 (2) 12565, 2024 doi: 10.4081/ejtm.2024.12565 upon Tyne, UK) was used to carry out quantitative label- free data analysis. Proteome Discoverer 2.2 using Sequest HT (Thermo Fisher Scientific) and a percolator were em- ployed for the identification of peptides and proteins. Da- tasets were imported into Progenesis QI software for further analysis. The multi-consensus MS file and listings of pro- teins in aged murine skeletal muscle that were generated by this proteomic study have been deposited under the title ‘Proteomic analysis of senescent mouse hindlimb muscles’ with the unique identifier ‘37txb’ to the Open Science Foun- dation (https://osf.io/37txb/). The standard bioinformatic analysis tools STRING (https://string-db.org) was used for the identification of potential protein-protein interaction patterns.57 Results The mass spectrometric analysis of crude tissue extracts from the aged mouse hindlimb musculature resulted in the identification of a large number of proteins involved in cel- lular signaling, bioenergetic metabolism and molecular chaperoning. The underlying objective of this investigation was to establish a proteomic reference map of aged skeletal muscles for future sarcopenia research. Proteomic profiling of proteins involved in the regulation of excitation-contraction coupling and calcium homeostasis in aged skeletal muscle The temporal and spatial fluxing of calcium ions through subcellular structures in myofibers is tightly regulated and plays a central role in the physiological facilitation of ex- citation-contraction coupling in skeletal muscles. Members of key protein complexes that are involved in the regulation of calcium homeostasis and second mes- senger signaling cascades were identified by mass spec- trometric screening of aged mouse hindlimb muscles, including i) the voltage-sensing L-type calcium channel complex of the transverse tubules (alpha-1S (voltage sen- sor), alpha-2/delta-1 and beta-1 subunits of the dihydro- pyridine receptor), ii) the calcium release channel complex of the triad junctions that is positioned between the transverse tubules and terminal cisternae region of the sarcoplasmic reticulum (ryanodine receptor calcium re- lease channel isoforms RYR1 and RYR2, and the auxiliary proteins triadin, junctophilin JPH1 and JPH2, and the junctional sarcoplasmic reticulum protein JSRP1), iii) lu- minal high-capacity calcium buffering proteins of the sar- coplasmic reticulum (calsequestrin isoforms fast CSQ1 and slow CSQ2, sarcalumenin), iv) calcium-pumping AT- Pase complexes involved in the fast re-uptake or removal of calcium from the sarcosol (sarcoplasmic reticulum cal- cium ATPases of the fast SERCA1 and slow SERCA2 type, as well as the plasma membrane calcium-transport- ing ATPase PMCA1), and v) additional calcium homeo- stasis regulators (parvalbumin, protein S100-A1, SH3 and cysteine-rich domain-containing protein STAC3, and stro- mal interaction molecule STIM1), and are listed in below Table 1. Excitation-contraction uncoupling is one of the mechanisms that is widely discussed to play a central role in the pathophysiological process that may lead to sar- copenia of old age. Findings from previous studies on muscle aging,45-50,58-65 imply reduced expression patterns and/or shifting to slower isoforms of calcium-handling proteins, such as the voltage sensor complex of transverse tubules, the calcium release channels of triad junctions, luminal and cytosolic calcium-binding proteins and cal- cium pumps of the sarcoplasmic reticulum. Proteomic profiling of proteins involved in major bioenergetic pathways in aged skeletal muscle Since sustained levels of contractile activity require a con- stant supply of energy in the form of ATP, the regulation and maintenance of bioenergetic pathways is of crucial impor- tance for the proper functioning of the neuromuscular sys- tem. The mass spectrometric analysis of aged mouse hindlimb muscles has identified a large number of enzymes involved in skeletal muscle energy metabolism. Table 2 lists major components that are involved in anaerobic glycolysis versus oxidative mitochondrial pathways. This includes pro- teins of the glycolytic pathway, gluconeogenesis and gly- cogen metabolism, as well as markers of the mitochondrial outer membrane, inner membrane and matrix that are essen- tial components of respiratory complexes I to V. Listed are also key metabolite transporters, such as fatty acid-binding proteins (FABP3, FABP4, FABP5), albumin and the oxygen transporter myoglobin (Table 2). The higher susceptibility of type II fibers to age-related muscular atrophy results in a fast-to-slow transition process, which is clearly reflected by isoform switching of major sarcomeric proteins, as recently reviewed.66 In analogy, age-associated fiber type shifting was shown to also affect the abundance of bioenergetic enzymes. Previously published reports on skeletal muscle aging,45-50,58-65 suggest indirect glycolytic-to-oxidative changes in energy metabolism in senescent myofiber pop- ulations. Decreased muscle proteins include key enzymes of glycolysis, such as pyruvate kinase and phosphofructo- kinase. In contrast, increased proteins of oxidative metabo- lism were identified as mitochondrial succinate dehydrogenase, ATP synthase and NADH dehydrogenase. Proteomic profiling of molecular chaperones involved in the cellular stress response in aged skeletal muscle The cellular stress response plays an essential role in the prevention of proteotoxic side effects in skeletal muscles. Both, the various classes of heat shock proteins and asso- ciated modulating enzymes are crucial factors that prevent abnormal protein folding and/or facilitate the swift removal of misfolded protein aggregates. Table 3 lists the mass spectrometric identification of key members of heat shock protein (HSP) classes HSPB/HSPE (small HSPs of 10-40 kDa) HSP40 (HSP70 co-chaperones, DNAJ), HSP60 (HSP70 co-chaperone, chaperonin), HSPA (HSP70s of ap- proximately 70 kDa), and HSPC/HSPH (large HSP90/ HSP110 of approximately 90 kDa), as well as calcium- binding chaperones of the luminal endoplasmic reticulum (calnexin and calreticulin/calregulin) and protein disulfide isomerase (PDI) in aged mouse hindlimb muscles. The proteomic profiling of the superfamily of peptidyl-prolyl cis-trans isomerases (PPIase) is shown in below Table 4. - 87 - Non -co mmerc ial us e o nly https://osf.io/37txb/ Proteomic reference map for sarcopenia research Part 2 Eur J Transl Myol 34 (2) 12565, 2024 doi: 10.4081/ejtm.2024.12565 - 88 - Table 1. Mass spectrometry-based proteomic profiling of proteins involved in the regulation of excitation-contrac- tion coupling and calcium homeostasis in aged mouse hindlimb muscle. Accession Protein name Gene Peptides Coverage Molecular mass (%) (kDa) (i) Transverse tubular voltage-sensing complex Q02789 Voltage-dependent L-type calcium Cacna1s 18 12.3 210.3 channel, alpha-1S O08532 Voltage-dependent calcium channel, Cacna2d1 30 39.6 124.6 alpha-2/delta-1 Q8R3Z5 Voltage-dependent L-type calcium Cacnb1 12 25.1 65.5 channel subunit beta-1 (ii) Triad junction calcium release complex E9PZQ0 Ryanodine receptor RYR1 Ryr1 157 46.0 565.0 E9Q401 Ryanodine receptor RYR2 Ryr2 9 2.4 564.8 E9Q9K5 Triadin Trdn 14 19.6 77.8 Q9ET80 Junctophilin JPH1 Jph1 10 19.7 71.9 Q9ET78 Junctophilin JPH2 Jph2 12 27.2 74.7 Q3MI48 Junctional sarcoplasmic reticulum Jsrp1 6 34.3 36.1 protein JSRP1 (iii) Luminal calcium buffering in sarcoplasmic reticulum O09165 Calsequestrin CSQ1, fast Casq1 16 60.7 46.4 O09161 Calsequestrin CSQ2, slow Casq2 10 42.4 48.2 Q7TQ48 Sarcalumenin Srl 24 62.5 54.3 (iv) Calcium removal via sarcoplasmic reticulum and sarcolemma Q8R429 Calcium ATPase SERCA1, fast Atp2a1 58 55.1 109.4 O55143 Calcium ATPase SERCA2, slow Atp2a2 47 48.7 114.9 G5E829 Plasma membrane Atp2b1 16 18.4 134.8 calcium-transporting ATPase PMCA1 (v) Calcium homeostasis regulation P32848 Parvalbumin Pvalb 17 84.5 11.9 P56565 Protein S100-A1 S100a1 2 29.8 10.5 Q8BZ71 SH3 and cysteine-rich Stac3 4 16.9 41.0 domain-containing protein STAC3 P70302 Stromal interaction molecule 1 Stim1 15 30.4 77.6 Non -co mmerc ial us e o nly Proteomic reference map for sarcopenia research Part 2 Eur J Transl Myol 34 (2) 12565, 2024 doi: 10.4081/ejtm.2024.12565 - 89 - Table 2. Mass spectrometry-based proteomic profiling of key proteins involved in major bioenergetic pathways in aged mouse hindlimb muscle. Accession Protein name Gene Peptides Coverage Molecular mass (%) (kDa) (i) Glycolysis, gluconeogenesis and glycogen metabolism P17710 Hexokinase-1 Hk1 19 21.5 108.3 O08528 Hexokinase-2 Hk2 32 43.6 102.5 P47857 Phosphofructokinase, muscle Pfkm 32 54.6 85.3 P05064 Fructose-bisphosphate aldolase Aldoa 37 89.3 39.4 ALDOA P17751 Triosephosphate isomerase Tpi1 13 67.9 26.7 P16858 Glyceraldehyde-3-phosphate Gapdh 24 80.8 35.8 dehydrogenase P09411 Phosphoglycerate kinase 1 Pgk1 37 84.2 44.6 Q9D0F9 Phosphoglucomutase-1 Pgm1 36 77.4 61.4 P17182 Alpha-enolase Eno1 28 79.5 47.1 P52480 Pyruvate kinase PKM Pkm 45 80.2 57.8 Q9R062 Glycogenin-1 Gyg1 11 49.2 37.4 Q9WUB3 Glycogen phosphorylase, muscle Pygm 53 62.6 97.3 Q9Z1E4 Glycogen synthase, muscle Gys1 21 40.9 83.9 (ii) Mitochondria Q60932 Voltage-dependent anion-selective Vdac1 21 76.0 32.4 channel protein 1 (mt-VDAC1; outer mitochondrial membrane) Q9DCS9 NADH dehydrogenase (inner Ndufb10 6 47.7 21.0 mitochondrial membrane complex I) Q9CQA3 Succinate dehydrogenase (inner Sdhb 16 55.3 31.8 mitochondrial membrane complex II) Q9DB77 Cytochrome b-c1 (inner Uqcrc2 17 49.4 48.2 mitochondrial membrane complex III) P56391 Cytochrome c oxidase (inner Cox6b1 5 61.6 10.1 mitochondrial membrane complex IV) P56480 ATP synthase subunit beta (inner Atp5b 37 79.2 56.3 mitochondrial membrane complex V) P54071 Isocitrate dehydrogenase Idh2 26 49.8 50.9 (mitochondrial matrix) (iii) Metabolite transportation P11404 Fatty acid-binding protein, heart Fabp3 8 64.7 14.8 P04117 Fatty acid-binding protein, adipocyte Fabp4 9 67.4 14.7 Q05816 Fatty acid-binding protein, epidermal Fabp5 4 30.4 15.1 P07724 Serum albumin Alb 48 73.7 68.7 P04247 Myoglobin Mb 10 77.3 17.1 Non -co mmerc ial us e o nly Proteomic reference map for sarcopenia research Part 2 Eur J Transl Myol 34 (2) 12565, 2024 doi: 10.4081/ejtm.2024.12565 - 90 - Table 3. Mass spectrometry-based proteomic profiling of molecular chaperones involved in the cellular stress re- sponse in aged mouse hindlimb muscle. Accession Protein name Gene Peptides Coverage Molecular mass (%) (kDa) (i) Small heat shock proteins (10-40 kDa, HSPB, HSPE) P14602 HSPB1 (Hsp27, beta-1) Hspb1 11 68.9 23.0 Q99PR8 HSPB2 (MKBP, beta-2) Hspb2 6 56.6 20.4 Q9QZ57 HSPB3 (b-3) Hspb3 2 26.0 17.2 P23927 HSPB5 (alphaB-Crystallin, alphaBC) Cryab 8 55.4 20.1 Q5EBG6 HSPB6 (Hsp20, beta-6) Hspb6 6 61.7 17.5 P35385 HSPB7 (cvHsp, beta-7) Hspb7 6 48.5 18.6 Q9JK92 HSPB8 (Hsp22; beta-8) Hspb8 5 25.5 21.5 Q64433 HSPE (10 kDa Hsp, mt) Hspe1 8 79.4 11.0 (ii) HSP40 heat shock proteins (HSP70 co-chaperones, DNAJ) P63037 DnaJ A1 HSP Dnaja1 5 22.9 44.9 Q9QYJ0 DnaJ A2 HSP Dnaja2 11 40.0 45.8 Q99M87 DnaJ A3 HSP Dnaja3 9 30.6 52.4 Q9JMC3 DnaJ A4 HSP Dnaja4 3 12.1 44.9 (iii) HSP60 heat shock proteins (HSP70 co-chaperone, chaperonin) P63038 HSPD1 (60 kDa Hsp, mt) Hspd1 55 75.0 61.0 (iv) HSP70 heat shock proteins (HSPA) Q61696 HSPA1A (70 kDa protein 1A, Hspa1a 14 27.0 70.1 Hsp72, inducible Hsp70) P17156 HSPA2 (heat shock-related 70 kDa Hspa2 28 45.0 69.6 protein 2, Hsp70-2) Q61316 HSPA4 (heat shock 70 kDa Hspa4 28 43.9 94.1 protein 4, Hsp-110) P20029 HspA5 (GRP78, BiP), ER Hspa5 64 73.4 72.4 P63017 HSPA8 (Hsc70, Hsp73, constitutive Hspa8 55 83.0 70.9 Hsp70) Q99M31 HSPA14 (heat shock 70 kDa Hspa14 2 5.9 54.7 protein 14, Hsp60) (v) HSP90/HSP110 large heat shock proteins (90 kDa, HSPC, HSPH) P07901 HSPC1 (Hsp90AA1, HSP90a) Hsp90aa1 47 64.7 84.9 P11499 HSPC3 (Hsp90AB1, HSP 90b) Hsp90ab1 67 80.7 83.3 P08113 HSPC4 (Hsp90B1, GRP94, Hsp90b1 70 67.0 92.5 endoplasmin), ER Q9CQN1 HSPC5 (75 kDa Hsp, mt) Trap1 27 36.7 80.2 Q61081 Hsp90 co-chaperone Cdc37 Cdc37 7 19.5 44.6 Q61699 HSPH1 (Hsp110) Hsph1 19 29.4 96.4 To be continued on next page Non -co mmerc ial us e o nly Proteomic reference map for sarcopenia research Part 2 Eur J Transl Myol 34 (2) 12565, 2024 doi: 10.4081/ejtm.2024.12565 - 91 - Table 3. Continued from previous page. Accession Protein name Gene Peptides Coverage Molecular mass (%) (kDa) (vi) Ca2+-binding chaperones of the luminal endoplasmic reticulum P35564 Calnexin (CNX) Canx 27 45.9 67.3 P14211 Calreticulin (CRT), calregulin Calr 24 72.4 48.0 (vii) Protein disulfide isomerase P27773 PDI A3 Pdia3 38 64.0 56.7 P08003 PDI A4 Pdia4 24 36.4 72.0 Q921X9 PDI A5 Pdia5 13 25.5 59.3 Q922R8 PDI A6 Pdia6 10 28.6 48.1 Table 4. Mass spectrometry-based proteomic profiling of the superfamily of peptidyl-prolyl cis-trans isomerases in aged mouse hindlimb muscle. Accession Protein name Gene Peptides Coverage Molecular mass (%) (kDa) P17742 PPIase PPIA Ppia 13 77.4 18.0 P24369 PPIase PPIB Ppib 14 55.1 23.7 P30412 PPIase PPIC Ppic 2 11.8 22.8 Q9CR16 PPIase PPID Ppid 16 42.2 40.7 Q9QZH3 PPIase PPIE Ppie 3 14.6 33.4 Q99KR7 PPIase PPIF, mt Ppif 7 39.3 21.7 A2AR02 PPIase PPIG Ppig 5 9.0 88.3 Q9D868 PPIase PPIH Ppih 3 14.9 20.5 P26883 PPIase FKBP1A Fkbp1a 9 79.6 11.9 P45878 PPIase FKBP2 Fkbp2 5 31.4 15.3 Q62446 PPIase FKBP3 Fkbp3 11 48.2 25.2 P30416 PPIase FKBP4 Fkbp4 17 38.0 51.6 O54998 PPIase FKBP7 Fkbp7 6 25.7 24.9 O35465 PPIase FKBP8 Fkbp8 6 20.4 43.5 Q9Z247 PPIase FKBP9 Fkbp9 14 27.5 63.0 Q61576 PPIase FKBP10 Fkbp10 11 30.3 64.7 Q9D1M7 PPIase FKBP11 Fkbp11 2 14.4 22.1 P59024 PPIase FKBP14 Fkbp14 3 16.1 24.3 Q9QUR7 PPIase NIMA-interacting PIN1 Pin1 3 29.7 18.4 Non -co mmerc ial us e o nly Proteomic reference map for sarcopenia research Part 2 Eur J Transl Myol 34 (2) 12565, 2024 doi: 10.4081/ejtm.2024.12565 Since skeletal muscle aging is associated with impaired mi- crocirculation, imbalanced proteostasis, mitochondrial dys- function, abnormal ion handling and stem cell exhaustion, a high degree of cellular and oxidative stress occurs in se- nescent fibers. Although the hierarchy within these different degenerating pathways has not yet been determined, it is clear from the characterization of aged muscles that a vari- ety of molecular chaperones are upregulated during sar- copenia.45-50,58-65 This includes a considerable number of large heat shock proteins, but especially drastically elevated levels of small heat shock proteins, such as alphaB-crystal- lin/HspB5 and the muscle-specific cardiovascular cvHsp/HspB7.65 Importantly, PPIases are intrinsically involved in the cellu- lar stress response in skeletal muscles. The PPIases mediate a rate-limiting step during protein folding by catalyzing the cis/trans-isomerization of the peptidyl-prolyl peptide bond. This is a crucial step of peptide synthesis that generates a properly folded and functional protein species. The members of this superfamily of immunophilins, that was identified by the mass spectrometric screening of aged mouse hindlimb muscles, are listed in Table 4. This detailed table of identified PPIases is included in this report to dem- onstrate the biochemical sensitivity of the streamlined bot- tom-up proteomic approach used to generate a reference map of proteins from crude extracts of senescent mouse muscles. Proteomic profiling of organellar and subcellular markers in aged skeletal muscle Robust marker proteins that are enriched in subcellular structures are extremely important for the swift cell biolog- ical characterization of changes in senescent skeletal mus- cles. Table 5 lists identified marker proteins of the sarcolemma, caveolae, ribosomes, the Golgi apparatus, per- oxisomes, proteasomes, lysosomes, the nucleus and cytosol in aged muscles. In addition, proteomic markers of the neu- romuscular junction, motor neurons and Schwann cells are listed in Table 5. Above tables have already covered pro- teomic markers of the transverse tubular membrane system, triad junctions and the sarcoplasmic reticulum. Bioinformatic analysis of potential protein-protein interactions within protein clusters in aged skeletal muscle Following mass spectrometric identification, muscle pro- teins were analyzed for potential protein interaction pat- terns. As shown in Figure 1, bioinformatic STRING analyses,57 indicates interaction patterns within protein clusters that are involved the regulation of excitation-con- traction coupling, calcium homeostasis and major bioener- getic pathways of aerobic and anaerobic metabolism, as well as the cellular stress response. Due to the complexity of functional and physical protein clustering and the large number of interacting protein species, the illustrations de- picted in Figure 1 are not presented to give detailed infor- mation on individual protein interactions, but instead are shown to provide a general overview of complex formation of identified protein groupings in aged skeletal muscles. Discussion Skeletal muscles contribute to approximately half of the biomass in the average human body.67 Voluntary muscles are involved in locomotion, posture, balance, bodily pro- tection, respiration, heat homeostasis and communication.68- 70 The range of muscular disorders ranges from genetic diseases of early childhood,71 to progressive muscle wasting syndromes in the elderly.72-76 It is therefore crucial to estab- lish proteomic reference maps of skeletal muscles at differ- ent age.77-82 This report focused on the establishment of the proteomic profile of the senescent mouse hindlimb muscu- lature. The proteomic databank can now be used as the sci- entific basis for detailed future studies into age-associated alterations in distinct muscle protein families. The proteomic analysis has covered important markers of organelles, such as the nucleus, Golgi apparatus, sar- colemma, caveolae, transverse tubules, triad junctions, sar- cosol, sarcoplasmic reticulum, mitochondria, lysosomes, proteasomes and peroxisomes. Metabolic adaptations of aged muscles can be studied at the level of both aerobic and anaerobic bioenergetic pathways. The proteomic strategy presented here has identified key markers for such studies, including enzymes that are present in major mitochondrial substructures.83 This is important, since mitochondrial ab- normalities and oxidative stress have been implicated in sar- copenia of old age.84,85 The identified mitochondrial markers encompass the voltage-dependent anion-selective channel VDAC1, NADH dehydrogenase, succinate dehydrogenase, cytochrome b-c1, cytochrome c oxidase, ATP synthase sub- unit beta and isocitrate dehydrogenase of the outer mem- brane, inner membrane complexes I to V and the mitochondrial matrix, respectively.86 The presence of gly- colytic enzymes and other components involved in glucose metabolism were clearly revealed by proteomics.87 Espe- cially interesting was the identification of both hexokinase isoforms, HKI and HKII, the enzymes that mediate the ini- tial priming step of muscle glycolysis and are differentially affected by hormonal regulation.88 Excitation-contraction coupling and calcium handling were previously shown to be impaired in aged skeletal muscles.89-91 The proteomic catalogue presented here lists all major players of this crucial signaling mechanism, in- cluding the voltage-sensing L-type calcium channel of the transverse tubules, the ryanodine receptor calcium release channel of the sarcoplasmic reticulum and its auxiliary complex at the triad junctions, as well as crucial regulatory proteins and luminal calcium binding proteins. This will enable future systems biological investigations to better determine the mechanisms that underlie excitation-con- traction uncoupling and associated muscular weakness in aged organisms.91 The expression levels of the protein constituents of the cellular stress response are highly abundant and diverse in skeletal muscles,92 and considerably affected during muscle adaptations and neuromuscular disease.93 The pro- teomic cataloguing of aged skeletal muscles reported in this article has listed a large number of molecular chap- erones and heat shock proteins. This included small heat shock proteins (HSPB/HSPE), which are present at high - 92 - Non -co mmerc ial us e o nly Proteomic reference map for sarcopenia research Part 2 Eur J Transl Myol 34 (2) 12565, 2024 doi: 10.4081/ejtm.2024.12565 - 93 - Table 5. Mass spectrometry-based proteomic profiling of organellar and subcellular markers in aged mouse hind- limb muscle. Accession Protein name Gene Peptides Coverage Molecular mass (%) (kDa) (i) Sarcolemma Q8VDN2 Na+/K+-ATPase, alpha-1 Atp1a1 34 38.7 113.0 Q6PIE5 Na+/K+-ATPase, alpha-2 Atp1a2 30 36.8 112.2 Q9WV27 Na+/K+-ATPase, alpha-4 Atp1a4 7 7.8 114.9 P14094 Na+/K+-ATPase, beta-1 Atp1b1 6 27.6 35.2 P14231 Na+/K+-ATPase, beta-2 Atp1b2 8 32.8 33.3 P97370 Na+/K+-ATPase, beta-3 Atp1b3 2 9.7 31.8 Q9ESD7 Dysferlin Dysf 27 18 237.9 Q69ZN7 Myoferlin Myof 34 21 233.3 P82348 Sarcoglycan, gamma Sgcg 2 11.3 32.1 (ii) Caveolae P49817 Caveolin-1 Cav1 12 68 20.5 Q9WVC3 Caveolin-2 Cav2 4 40.7 18.2 P51637 Caveolin-3 Cav3 3 25.8 17.4 (iIi) Organellar markers (ribosome, Golgi apparatus, peroxisome, proteasome, lysosome) P14206 40S ribosomal protein SA (ribosome) Rpsa 18 63.1 32.8 P55937 Golgin-160 (Golgi apparatus) Golga3 6 6.0 167.2 P24270 Catalase (peroxisome) Cat 30 63.9 59.8 P61089 Ubiquitin-conjugating enzyme E2 Ube2n 8 55.3 17.1 (proteasome) P11438 Lysosome-associated membrane Lamp1 3 7.6 43.9 glycoprotein 1 (lysomsome) (iv) Nucleus O08579 Emerin (inner nuclear membrane) Emd 5 28.2 29.435 P48678 Lamin-A/C (nuclear lamina) Lmna 60 73.7 74.2 P21619 Lamin-B2 (nuclear lamina) Lmnb2 15 24.8 67.3 P10922 Histone H1.0 H1f0 5 27.3 20.9 Q64522 Histone H2A Hist2h2ab 8 66.2 14.0 P02301 Histone H3.3 H3f3c 7 47.1 15.3 P62806 Histone H4 Hist1h4a 12 60.2 11.4 To be continued on next page Non -co mmerc ial us e o nly Proteomic reference map for sarcopenia research Part 2 Eur J Transl Myol 34 (2) 12565, 2024 doi: 10.4081/ejtm.2024.12565 density in myofibers,65,94,95 the group of co-chaperoning Hsp40 and Hsp60 proteins, the highly diverse Hsp70 (HSPA) family of constitutive and inducible chaperoning proteins,96 the Hsp90 (HSPC/HSPH) class of large heat shock proteins,97 protein disulfide isomerases, calcium- binding chaperones and the superfamily of PPIases that catalyze the cis/trans-isomerization of the peptidyl-prolyl peptide bond during protein folding.98,99 The newly presented proteomic map of muscle-associated proteins in senescent mice can now be employed for the systematic evaluation of complex changes and adaptations during age-related muscle degeneration. This could have significant implications for the establishment of new pro- teomic biomarker signatures of aging.78,79,100,101 Hence, the new MS-based reference map could be useful for detailed future evaluations of (i) muscle fiber type shifting,33,66,102 (ii) metabolic disturbances and alterations of the gut-mus- cle axis,103 (iii) mitochondrial dysfunction, inflammation and impaired cellular signaling,84 and (iv) protein mod- ifications,6 as well as the potential influence of (v) phar- macological therapies and related treatments,74,75,104 (vi) physical activity levels,105,106 (vii) nutritional interventions,104,107-109 and (viii) gender specific differ- ences,110 on the development of different forms of sarcope- nia.71-73 Hence, novel proteomic biomarkers could be beneficial for better defining and differentiating the pre- sarcopenic loss of skeletal muscle mass and decline in contractile function,111,112 from acute versus chronic forms of sarcopenia of old age.113,114 The quality of life can be severely impacted by the progres- sive loss of muscle mass and accompanying dysfunction of the skeletal musculature in the elderly. In order to better un- derstand the multi-factorial etiology of age-related muscle wasting, it is imperative to determine in more detail the mo- lecular mechanisms that trigger myofiber degeneration in the senescent organism. Animal models of aging play a crit- ical role in sarcopenia research.100,115,116 This report has out- lined the mass spectrometric analysis of total protein extracts from crude muscle tissue extracts, which has re- sulted in the establishment of a proteomic reference map of the aged mouse hindlimb musculature. Figure 2 gives an overview of identified protein groupings in senescent skele- tal muscles using bottom-up proteomics. The established protein clusters that are associated with the contractile apparatus in sarcomeres, various organ- elles, the excitation-contraction coupling apparatus, the extra-sarcomeric cytoskeleton, the extracellular matrix, bioenergetic pathways, the molecular chaperoning system and the nerve-muscle connection, are crucial entities for normal physiological functioning and efficient adaptabil- ity of the neuromuscular system. Many of these protein families are affected in sarcopenia of old age and are therefore crucial targets for studying the cellular mech- anisms that cause the age-related loss in skeletal muscle - 94 - Table 5. Continued from previous page. Accession Protein name Gene Peptides Coverage Molecular mass (%) (kDa) (v) Cytosol P13634 Carbonic anhydrase CA1 Ca1 2 10 28.3 P00920 Carbonic anhydrase CA2 Ca2 3 22.7 29.0 P16015 Carbonic anhydrase CA3 Ca3 17 85 29.4 P06151 Lactate dehydrogenase Ldha 23 74.4 36.5 P09528 Ferritin heavy chain Fth1 10 62.1 21.1 P29391 Ferritin light chain 1 Ftl1 6 48.6 20.8 (vi) Neuromuscular junction, motor neuron and Schwann cells P13595 Neural cell adhesion molecule Ncam1 10 12.4 119.4 NCAM1 P27573 Myelin protein P0 Mpz 5 20.2 27.6 P04370 Myelin basic protein Mbp 4 17.6 27.2 Q9CQW1 Synaptobrevin Ykt6 3 15.2 22.3 O09117 Synaptophysin Sypl1 2 13 28.9 Non -co mmerc ial us e o nly Proteomic reference map for sarcopenia research Part 2 Eur J Transl Myol 34 (2) 12565, 2024 doi: 10.4081/ejtm.2024.12565 - 95 - Figure 1. Overview of potential protein-protein interactions within major protein complexes that were detected by bot- tom-up proteomics of total extracts from aged mouse skeletal muscle. The data used to generate the individual images are the listings of identified protein families that are linked to calcium homeostasis and the regulation of excitation-contraction coupling (Table 1), bioenergetic pathways and metabolite transportation (Table 2), and molecular chaperones involved in the cellular stress response (Tables 3 and 4). The publicly available bioinformatic analysis tool STRING (https://string- db.org) was used for the analysis of protein interaction patterns.57 Non -co mmerc ial us e o nly Proteomic reference map for sarcopenia research Part 2 Eur J Transl Myol 34 (2) 12565, 2024 doi: 10.4081/ejtm.2024.12565 mass and contractile strength.45-50,58-65 The proteomic ref- erence map presented in this article covers the main con- stituents involved in the regulation and maintenance of the excitation-contraction-relaxation cycle and its structural embedding in the intracellular cytoskeleton and provision of lateral force transmission through costameres and the extracellular matrix. Building on these findings, tissue proteomics can now be used to routinely study small amounts of starting material for the in-depth screening of age-associated processes, such as excitation-contraction uncoupling, myonecrosis, cytoskeletal collapse, an altered cellular stress response, myofiber type shifting, abnormal calcium homeostasis, impaired bioenergetics, contractile weakness and reactive myofibrosis. Hence, the establishment of the proteomic profile of senescent muscles can be utilized as the basis of biomedical knowledge for detailed future investigations into age-related changes and/or adaptations of distinct pro- tein families. List of abbreviations BiP, Binding immunoglobulin protein CA, Carbonic anhydrase CAM, Neural cell adhesion molecule cv, cardiovascular CSQ, Calsequestrin ER, Endoplasmic reticulum FABP, Fatty acid binding protein FASP, Filter assisted sample preparation FKBP, FK506 binding protein GRP, Glucose-regulated protein Hsc, Heat shock cognate HSP, Heat shock protein JPH, Junctophilin JSRP, Junctional sarcoplasmic reticulum protein MKBP, Myotonic dystrophy protein kinase binding protein MS, Mass spectrometry mt, mitochondrial NIMA, ‘never in mitosis gene a’ kinase OSF, Open Science Foundation PDI, Protein disulfide isomerase. PMCA, Plasma membrane calcium-transporting ATPase PPIase, Peptidyl-prolyl cis/trans isomerases RYR, Ryanodine receptor SERCA, Sarcoplasmic or endoplasmic reticulum calcium ATPase SL, Sarcolemma SR, Sarcoplasmic reticulum STAC3, SH3 and cysteine-rich domain-containing protein TT, Transverse tubules VDAC, voltage-dependent anion-selective channel Contributions PD, DS and KO were involved in the conceptualization and initiation of this project, as well as the design of the research strategy. SG, MZ and PD were involved in the preparation of muscle tissues and performed the biochem- ical experiments and analyzed the data. MH and PM per- - 96 - Figure 2. Overview of mass spectrometrically identified protein groupings in aged mouse hindlimb muscles. Non -co mmerc ial us e o nly Proteomic reference map for sarcopenia research Part 2 Eur J Transl Myol 34 (2) 12565, 2024 doi: 10.4081/ejtm.2024.12565 formed the mass spectrometric and bioinformatic analysis. All authors were involved in the writing and final editing of the manuscript. Funding This work was supported by the Kathleen Lonsdale Institute for Human Health Research at Maynooth University. The Orbitrap Fusion Tribrid mass spectrometer was funded under a Science Foundation Ireland Infrastructure Award to Dublin City University (SFI 16/RI/3701). Conflict of interest The authors declare no competing interests. Ethics approval Local governmental and institutional animal care regula- tions were followed and approved by the Institutional Ani- mal Care and Use Committee (Amt für Umwelt, Verbraucherschutz und Lokale Agenda der Stadt Bonn, North Rhine-Westphalia, Germany). Transportation and usage of tissue specimens was in accordance with the reg- ulations of the Department of Agriculture (animal by-pro- duct register number 2016/16 to the Department of Biology, National University of Ireland, Maynooth). The study con- forms with the Helsinki Declaration of 1964, as revised in 2013, concerning human and animal rights. Availability of data and materials All data generated or analyzed during this study are in- cluded in this published article. Corresponding Author Kay Ohlendieck, Department of Biology, Maynooth Uni- versity, National University of Ireland, Maynooth W23F2H6, Co. Kildare, Ireland. Tel.: 353.1.7083842. ORCID ID: 0000-0002-6266-4510 E-mail: kay.ohlendieck@mu.ie Paul Dowling ORCID ID: 0000-0002-9290-9267 paul.dowling@mu.ie Stephen Gargan ORCID ID: 0000-0003-1005-8372 stephen.gargan@mu.ie Margit Zweyer ORCID ID: 0000-0001-5615-1869 margit.zweyer@dzne.de Michael Henry ORCID ID: 0000-0001-5312-4961 michael.henry@dcu.ie Paula Meleady ORCID ID: 0000-0001-5306-310X paula.meleady@dcu.ie Dieter Swandulla ORCID ID: 0000-0003-0923-7090 swandulla@uni-bonn.de References 1. Aebersold R, Mann M. Mass-spectrometric explora- tion of proteome structure and function. 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J Cachexia Sarcopenia Muscle 2021;12:538-54. - 100 - Non -co mmerc ial us e o nly Proteomic reference map for sarcopenia research Part 2 Eur J Transl Myol 34 (2) 12565, 2024 doi: 10.4081/ejtm.2024.12565 Disclaimer All claims expressed in this article are solely those of the authors and do not necessarily represent those of their af- filiated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher. Submitted: 12 April 2024. Accepted: 12 April 2024. Early access: 24 May 2024. - 101 - Non -co mmerc ial us e o nly