Layout 1 Thematic Section: Advances in Musculoskeletal and Neuromuscular Rehabilitation | Maccarone & Masiero Eur J Transl Myol 34 (2) 12564, 2024 doi: 10.4081/ejtm.2024.12564 In the matured human body, voluntary muscle fibers represent the most abundant cellular contributor to biomass,1 and are intrinsically involved in generating contractile force for maintaining proper fitness levels and health status.2-4 A high degree of skeletal muscle plasticity underlies the adaptative potential of the body to manage altered physiological demands and biochemical alterations, as well as fluctuations in metabolic and bioenergetic requirements,5 emphasizing the importance of mobility medicine for supporting optimized body movements.6 Skeletal muscle functions are diverse and include the promotion of coordinated locomotion via tightly controlled excitation-contraction-relaxation cycles, postural support to maintain muscle-skeletal balance and diaphragm-associated respiration.3 Furthermore, skeletal muscle contractions are involved in the regulation of body thermogenesis, bioenergetic integration and metabolic adaptations, as well as facial and verbal communication.4 Key questions of basic and applied myology are concerned with the molecular and cellular mechanisms that underlie the response of the voluntary contractile system to stressful stimuli such as pathological insults, extreme physical strain, disuse-related atrophy and aging, and how muscle fiber dys- function affects whole-body physiology.7 Many of these physiological and pathophysiological issues are studied with the help of animal models. In general, animal models play a central role in the biomedical sciences,8 including mice which are widely used in the neurosciences.9 Various mouse tissues are routinely studied to improve our biolog- ical knowledge of development, differentiation, maturation, physiological adaptations, environmental effects, heat ho- meostasis, dietary requirements and disease mechanisms,10- Abstract Sarcopenia of old age is characterized by the progressive loss of skeletal muscle mass and concomitant decrease in contractile strength. Age-related skeletal muscle dysfunctions play a key pathophysiological role in the frailty syndrome and can result in a drastically diminished quality of life in the elderly. Here we have used mass spectrometric analysis of the mouse hindlimb musculature to establish the muscle protein constellation at advanced age of a widely used sarcopenic animal model. Proteomic results were further analyzed by systems bioinformatics of voluntary muscles. In this report, the proteomic survey of aged muscles has focused on the expression patterns of proteins involved in the contraction-relaxation cycle, membrane cytoskeletal maintenance and the formation of the extracellular matrix. This includes proteomic markers of the fast versus slow phenotypes of myosin-containing thick filaments and actin- containing thin filaments, as well as proteins that are associated with the non-sarcomeric cytoskeleton and various matrisomal layers. The bioanalytical usefulness of the newly established reference map was demonstrated by the comparative screening of normal versus dystrophic muscles of old age, and findings were verified by immunoblot analysis. Key Words: aging, mass spectrometry, muscle proteomics, muscular dystrophy. Eur J Transl Myol 34 (2) 12564, 2024 doi: 10.4081/ejtm.2024.12564 Proteomic reference map for sarcopenia research: mass spectrometric identification of key muscle proteins located in the sarcomere, cytoskeleton and the extracellular matrix 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. - 66 - Non -co mmerc ial us e o nly Proteomic reference map for sarcopenia research Part 1 Eur J Transl Myol 34 (2) 12564, 2024 doi: 10.4081/ejtm.2024.12564 13 as well as the natural aging process.14-16 This includes mouse models of skeletal muscle aging.17-19 Skeletal muscles are majorly affected during aging, now generally referred to as sarcopenia of old age,20 and this pro- cess is characterized by the progressive loss of tissue mass and contractile strength.21-23 Sarcopenia can result in funda- mental consequences for the quality of life of aged individ- uals, since voluntary muscles are involved in a plethora of physiological functions in the body.24 Unbiased and tech- nology-driven approaches to carry out systematic surveys of aged tissues include omics-type techniques, such as ge- nomics, transcriptomics, proteomics, metabolomics and cy- tomics.25-27 Protein populations in senescent muscles can be conveniently determined by mass spectrometry-based pro- teomics.28-31 Skeletal muscle tissues from a variety of species have been extensively characterized by mass spectrometry using both bottom-up and top-down proteomics.32-35 The combined biochemical cataloguing efforts of numerous mass spectro- metric screening initiatives,36-39 have been instrumental to establish an ever-growing catalog of proteoforms that con- stitute the dynamic skeletal muscle proteome.40-42 Important studies in muscle proteomics have included the systematic analysis of fiber type specification,43-46 fiber type shifting,47- 49 the effects of exercise,50-53 the impact of primary muscle disease,54-56 neuromuscular dysfunction due to co-morbid- ities,57-59 and sarcopenia of old age.60-65 Building on these findings, we describe in this report the mass spectrometric profiling of total tissue extracts derived from the senescent mouse hindlimb musculature using bot- tom-up proteomics. The age of mouse models is usually categorized as ‘mature adult’ (3-6 months), ‘middle-aged’ (10-15 months) and ‘old’ (18-24 months).66 Early effects of natural aging can already be observed in the middle-aged group, but severe alterations, such as a drastic decline in muscle mass and progressive dysfunction, are only ob- served in the senescent group.15,67 Therefore, we have used 24 months old mouse skeletal muscle specimens for our proteomic mapping of the aged musculature. Materials and Methods Materials The proteomic analysis of mouse hindlimb muscles was carried out with analytical grade chemicals from Sigma Chemical Company (Dorset, UK), GE Healthcare (Little Chalfont, Buckinghamshire, UK) and Bio-Rad Laboratories (Hemel-Hempstead, Hertfordshire, UK). Trypsin (MS- grade), protease inhibitors (cOmplete™, mini protease in- hibitor cocktail) and spin filters (Vivacon 500, VN0H22; 30,000 MWCO) were obtained from ThermoFisher Scien- tific (Dublin, Ireland), Roche (Mannheim, Germany) and Sartorius (Göttingen, Germany), respectively. Protein as- says were carried out with the Pierce 660 nm Protein Assay Reagent from ThermoFisher Scientific (Dublin, Ireland). For protein gel electrophoresis and immunoblot analysis, precast Invitrogen Bolt 4-12% Bis-Tris gels and Whatman nitrocellulose transfer membranes were purchased from Bio-Science Ltd. (Dun Laoghaire, Ireland). InstantBlue Coomassie Protein Stain was from Expedeon (Heidelberg, Germany). For immunoblotting, primary antibodies were obtained from ThermoFisher Scientific, Dublin, Ireland (mAb SD83-03 against collagen VI), Merck Life Science Ltd, Arklow, Ireland (mAb PARV-19 against parvalbumin), R&D Systems, Minneapolis, MN, USA (MAB5718 against glyceraldehyde-3-phosphate dehydrogenase), and Abcam, Cambridge, UK (mAb ERP5158 against tropomyosin TPM1, alpha). Secondary peroxidase-conjugated anti-IgG were from Sigma Chemical Company (Dorset, UK). The visualization of immuno-decorated bands was performed by the enhanced chemiluminescence (ECL) technique using an ECL kit from Roche (Mannheim, Germany). Mouse 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).39 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).68 The mass spectrometric analysis of total skeletal muscle preparations was performed with protein extracts from 6 wild type mice. For the comparison of aged wild type versus aged and dystrophic mdx-4cv muscles, which lack the membrane cytoskeletal protein dystrophin,39 24 months old muscles were used. Protein extraction from skeletal muscle specimens The bottom-up proteomic analysis of hindlimb muscles from wild type C57BL6 mice, as well as age-matched dys- trophic mdx-4cv mice, was carried out with total protein ex- tracts.69 Tissue preparation, homogenization using pulverization of specimens via grinding in liquid nitrogen and protein extraction were performed by a standardized procedure.70 Following homogenization in lysis buffer (0.1 M dithiothreitol, 4% (w/v) sodium dodecyl sulfate, 100 mM Tris-Cl, pH 7.6; supplemented with a protease inhibitor cocktail), suspensions were briefly treated in a sonicating water bath and then heated for 3 min at 95oC.71 Samples were centrifuged at 16,000×g for 5 minutes and the protein- containing supernatant extracted for subsequent mass spec- trometric analysis. The protein concentration was determined with the help of the Pierce 660 nm Protein Assay system.72 For proteomics, samples were mixed with 8 M urea, 0.1 M Tris, pH 8.9 in Vivacon 500 spin filter units.73 Following a centrifugation step at 14,000×g for 15 min, the further processing of samples, the switching of buffers and peptide generation by trypsin digestion was car- - 67 - Non -co mmerc ial us e o nly Proteomic reference map for sarcopenia research Part 1 Eur J Transl Myol 34 (2) 12564, 2024 doi: 10.4081/ejtm.2024.12564 ried out by the filter-aided sample preparation (FASP) tech- nique, as described in detail by Wiśniewski.74 Mass spectrometry-based proteomic analysis A Thermo Orbitrap Fusion Tribrid mass spectrometer from Thermo Fisher Scientific (Waltham, MA, USA) was used for the label-free liquid chromatography mass spectrometric analysis of aged hindlimb muscles from normal mice (n=6 biological repeats; n=2 technical repeats), as well as the comparison of wild type versus dystrophic mdx-4cv mus- cles (n=3 biological repeats; n=2 technical repeats).56 The individual analytical steps (including FASP-based sample preparation, buffer composition, timing of preparative stages, data-dependent acquisition, and bioinformatic data handling) that were employed in the proteomic analysis, are described in detail in a recently published methods paper.75 A Thermo UltiMate 3000 nano system was used for re- versed-phase capillary high-pressure liquid chromatogra- phy and directly coupled in-line with the Thermo Orbitrap Fusion Tribrid mass spectrometer. The qualitative data anal- ysis 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 Percolator. Parameters for the mass spectrometric detection of senescent hindlimb muscle pro- teins 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 probability 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 Pro- genesis QI for Proteomics (version 2.0; Nonlinear Dy- namics, a Waters company, Newcastle upon Tyne, UK) was used to carry out quantitative label-free data analysis. Pro- teome Discoverer 2.2 using Sequest HT (Thermo Fisher Scientific) and a percolator were employed for the identifi- cation of peptides and proteins. Datasets were imported into Progenesis QI software for further analysis. The multi-consensus MS file and listings of proteins in aged murine skeletal muscle that were generated by this pro- teomic study have been deposited under the title ‘Proteomic analysis of senescent mouse hindlimb muscles’ with the unique identifier ‘37txb’ to the Open Science Foundation (OSF) (https://osf.io/37txb/). The standard bioinformatic analysis tools PANTHER (http://www.pantherdb.org) and STRING (https://string- db.org) were used for the identification of protein classes,76 and potential protein-protein interaction patterns,77 respectively. Immunoblot analysis Comparative immunoblotting of preparations from aged wild type versus aged mdx-4cv mouse hindlimb muscle was carried out by an optimized procedure, as previously de- scribed in detail.39 Protein separation was performed with precast Invitrogen Bolt 4-12% Bis-Tris gels. Separated pro- tein bands were visualized with InstantBlue Coomassie pro- tein stain. Following electrophoretic transfer onto Whatman nitrocellulose transfer membranes,68 individual blots were labelled with 1:1000 diluted primary antibodies to glyceral- dehyde 3-phosphate dehydrogenase, tropomyosin, parval- bumin or collagen isoform COL-VI. Membranes were washed and incubated with 1:1000 diluted secondary per- oxidase-conjugated antibodies. Washed membranes were then treated with enhanced chemiluminescence (ECL) chemicals to visualize immuno-decorated protein bands. Results The findings from the mass spectrometric screening of total tissue extracts from the aged mouse hindlimb musculature are presented in this report. The major aim of this study was to establish a proteomic reference map of aged skeletal muscles for future sarcopenia research. The focus was on the expression patterns of key proteins located in the sar- comere, non-sarcomeric cytoskeletal networks and the ex- tracellular matrix. To illustrate the bioanalytical value of the new proteomic data set, the mass spectrometric comparison of normal versus dystrophic skeletal muscles is presented, including comparative immunoblotting. Mass spectrometric analysis of aged mouse hindlimb muscle extracts Crude protein extracts from 100 mg muscle specimens were used as starting material to establish the optimum utilization of small tissue biopsy specimens. In order to minimize the number and complexity of preparative steps, the experi- mental protocol was conducted in a simple and streamlined way. The analysis pipeline used here represents a swift, eco- nomical and efficient proteomic procedure that avoids the introduction of excess bioanalytical artefacts and can be employed for the routine detection of key protein species in aged skeletal muscles, as shown by the results presented in below sections. All individual steps involved in the pro- teomic analysis pipeline, including a description of nec- essary materials, chemicals, buffers and the critical timing of preparative stages, as well as data-dependent acquisition and bioinformatic data handling, have been described in de- tail in a recently published methods paper.78 The proper planning of experimental protocols, the optimized prepara- tion of buffers and the continues maintenance of equipment are essential for the successful execution of a proteomic analysis. Streamlined sample processing can considerably reduce overall running costs and prevent the introduction of analytical artefacts.32,75 The proteomic profiling of the senescent mouse hindlimb musculature detected 3128 distinct protein species with 2 or more peptides. The multi-consensus MS file and listings of proteins in aged skeletal muscle that were generated by proteomics as outlined in this report have been deposited under the title ‘Proteomic analysis of senescent mouse hind- limb muscles’ with the unique identifier ‘37txb’ to the OSF site (https://osf.io/37txb/). The results are based on multi- consensus data from 6 biological repeats and 2 technical re- peats. The muscle-associated protein with the lowest molecular mass was identified as the actin-sequestering - 68 - Non -co mmerc ial us e o nly https://osf.io/37txb/ https://osf.io/37txb/ Proteomic reference map for sarcopenia research Part 1 Eur J Transl Myol 34 (2) 12564, 2024 doi: 10.4081/ejtm.2024.12564 protein thymosin beta-4 of 5.7 kDa and that with the highest molecular mass as the abundant sarcomeric protein titin of 3906.4 kDa. Besides the giant half-sarcomere spanning pro- tein titin, other identified high-molecular-mass muscle pro- teins above 400 kDa included laminin, dystrophin, ankyrin-2, sacsin, dynein, plectin, the ryanodine receptor calcium release channel, epiplakin, nesprin, dystonin and obscurin. As shown in Figure 1, the total number of peptides per de- tected protein ranged from 2 to 1807. The diagrams show the number of peptides of the top 1-20, top 21-100 and top 101-3128 detected protein species. An amino acid sequence coverage based on more than 120 individual peptides included titin, plectin, myosin-4, myosin-1, ob- scurin, ryanodine receptor isoform RYR1, myosin-8, myo- sin-7, dynein DYNC1H1 and myosin-9. Sequence coverage of identified proteins ranged from 0.4% (E3 ubi- quitin-protein ligase RNF213 of 584.3 kDa) to 95.5% (cy- toplasmic actin ACTG1 of 41.8 kDa). A sequence coverage above 90% was achieved in the case of cytoplas- mic actin 1 and 2, skeletal muscle-specific myosin regu- latory light chain MLC2/MYLPF, the 60S acidic ribosomal protein RPLP2, guanine nucleotide-binding protein subunit beta-2, dolichyl-diphospho-oligosaccha- ride-protein glycosyl-transferase subunit 4, peroxiredoxin- 1, phosphatidyl-ethanolamine-binding protein PEBP1 and fructose-bisphosphate aldolase ALDOA. Bioinformatic PANTHER analysis of protein families in aged skeletal muscle The bioinformatic analysis tool PANTHER,76 was used to generate a pie chart that displays the main types of identified protein families in aged skeletal muscles. As shown in Figure 2, the most abundantly identified mus- cle-associated proteins belong to the classes of metabolite interconversion enzymes, protein modifying enzymes, RNA metabolism proteins, cytoskeletal proteins, mem- brane traffic proteins, scaffold/adaptor proteins, trans- porters and translational proteins. In addition, skeletal muscle protein classes were identified as DNA metabo- lism proteins, calcium-binding proteins, cell adhesion molecules, cell junction proteins, molecular chaperones, chromatin, chromatin-binding proteins, defence proteins, immunity proteins, extracellular matrix proteins, gene- specific transcriptional regulators, intercellular signal molecules, protein-binding activity modulators, storage proteins, structural proteins, transfer proteins, carrier pro- teins and transmembrane signal receptors. Proteomic profiling of sarcomeric proteins and contractile apparatus in aged skeletal muscle Sarcomeres containing the contractile acto-myosin appa- ratus and auxiliary filaments, in conjunction with the extra- sarcomeric cytoskeletal network, constitute more than half - 69 - Figure 1. Overview of the proteomic mapping of mouse skeletal muscle extracts. Non -co mmerc ial us e o nly Proteomic reference map for sarcopenia research Part 1 Eur J Transl Myol 34 (2) 12564, 2024 doi: 10.4081/ejtm.2024.12564 of the protein mass in voluntary muscles. The proteomic analysis of aged skeletal muscle clearly identified the main protein species that are associated with these subcellular structures that are involved in filament sliding and intra- cellular stabilization, including i) the myosin-containing thick filaments (myosin heavy chains, myosin light chains, myosin binding proteins), ii) the actin-containing thin fil- aments (muscle and heart actins) in combination with the troponin-tropomyosin regulatory complex (troponin sub- units TnC, TnT and TnI, as well as tropomyosin TPM) and auxiliary proteins (F-actin-capping proteins, tropomod- ulins, Xin actin-binding protein and myopalladin), iii) the half-sarcomere spanning titin filaments (titin and the titin- associated muscle ankyrin repeat protein MARP), iv) the sarcomeric M-line complex (obscurin, myomesin), and v) the sarcomeric Z-line complex (alpha-actinin, filamin-C, telethonin and myozenin; Table 1). Of note, the types of myosin light and heavy chains ranged from major slow and fast forms to developmental isoforms. This included myosin heavy chains (MyHC) of the fast type (MyHC-IIb, MyHC-IIx) and their slower counterparts (MyHC-I, MyHC-7b) and the mostly developmental types MyHC-embryonic and MyHC-perinatal. Myosin light chains (MLC) were identified as MLC1/MLC3 and mus- cle-specific MLC2, as well as the slow chains MLC2 and MLC3. In analogy, the regulatory troponin (Tn) subunits TnC, which is modified by calcium binding, TnT, which binds to tropomyosin, and TnI, which is involved in the in- hibitory mechanism of acto-myosin interactions, were found to be present in both slow and fast isoforms. Tropo- myosin was represented by its isoforms TPM1, TPM3 and TPM4 (Table 1). Previous studies of aged human muscles and animal models of sarcopenia have shown considerable changes in these identified sarcomeric proteins during the aging process,60-65,79-86 indicating age-related fiber type shift- ing.30 Graded muscle transformations involve a general trend of isoform transitions from faster types of MyHC and MLC, as well as TnC, TnI and TPM to their slower coun- terparts.80,82,84 Thus, the proteomic catalog of aged muscle presented here can provide a suitable overview of protein isoform expression patterns that can be related to other aging studies and be highly useful as a reference databank for studying proteome-wide changes in senescent myofiber populations. As recently reviewed,30 age-associated in- creases in distinct isoforms of sarcomeric proteins include MyHC-I, MLC-2s, MLC-1/3f, ACTA, TNT-3, MYOZ-1, MARP/ANKRD2, ACTC, and concomitant decreases were shown for MyHC-IIx, MLC-1/3, MLC2f, TnT-1, TnT-3, TnC-1, TPM-3, FLNC and TTN, as well as shifting of phosphorylated MLC-2f to MLC-2s isoforms. - 70 - Figure 2. Overview of protein families detected by the bottom-up proteomic analysis of total extracts from aged mouse skeletal muscle. The publicly available bioinformatic analysis tool PANTHER (http://www.pantherdb.org) was used for the identification of protein classes.76 Non -co mmerc ial us e o nly http://www.pantherdb.org Proteomic reference map for sarcopenia research Part 1 Eur J Transl Myol 34 (2) 12564, 2024 doi: 10.4081/ejtm.2024.12564 - 71 - Table 1. Mass spectrometry-based proteomic profiling of sarcomeric proteins in aged mouse hindlimb muscle. Accession Protein name Gene Peptides Coverage Molecular mass (%) (kDa) (i) Half-sarcomere spanning titin filament A2ASS6 Titin Ttn 1807 63.2 3906.4 Q9WV06 Titin-associated MARP Ankrd2 10 42.1 36.7 (ii) Sarcomeric M-line complex A2AAJ9 Obscurin Obscn 163 35.0 966.6 Q62234 Myomesin-1 Myom1 100 68.5 185.5 A2ABU4 Myomesin-3 Myom3 27 27.0 161.8 (iii) Sarcomeric Z-line complex Q7TPR4 Alpha-actinin-1 Actn1 55 70.0 103.1 Q9JI91 Alpha-actinin-2 Actn2 53 63.9 103.8 O88990 Alpha-actinin-3 Actn3 64 75.2 103.0 P57780 Alpha-actinin-4 Actn4 56 69.5 105.0 Q8VHX6 Filamin-C, muscle Flnc 100 51.6 291.1 O70548 Telethonin Tcap 8 55.1 19.1 Q9JK37 Myozenin-1 Myoz1 15 72.6 31.5 Q9JJW5 Myozenin-2 Myoz2 5 28.4 29.8 Q8R4E4 Myozenin-3 Myoz3 9 45.3 27.0 (iii) Thick filament myosin complex Q5SX39 Myosin MyHC-IIb, fast Myh4 200 73.1 222.9 Q5SX40 Myosin MyHC-IIx, fast Myh1 179 70.3 223.3 Q91Z83 Myosin MyHC-I(beta), slow Myh7 126 56.9 222.9 A2AQP0 Myosin MyHC-7b, slow Myh7b 20 6.7 221.5 P13541 Myosin MyHC-embryonic Myh3 73 29.3 223.8 P13542 Myosin MyHC-perinatal Myh8 133 50.0 222.7 P05977 Myosin MLC1/MLC3 Myl1 23 88.8 20.6 P97457 Myosin MLC2, muscle Mylpf 18 92.9 19.0 P51667 Myosin MLC2, slow Myl2 9 64.5 18.9 P09542 Myosin MLC3, slow Myl3 15 77.5 22.4 Q8VCR8 Myosin light chain kinase 2 Mylk2 12 35.6 66.0 Q5XKE0 Myosin-binding protein MyBP-C Mybpc2 73 71.8 127.4 P70402 Myosin-binding protein MyBP-H Mybph 21 66.5 52.6 To be continued on next page Non -co mmerc ial us e o nly Proteomic reference map for sarcopenia research Part 1 Eur J Transl Myol 34 (2) 12564, 2024 doi: 10.4081/ejtm.2024.12564 Proteomic profiling of non-sarcomeric cytoskeletal proteins in aged skeletal muscle Major extra-sarcomeric cytoskeletal proteins are listed in Table 2, including i) the major cytolinker component plec- tin, ii) adaptor proteins such as ankyrin, iii) membrane cy- toskeletal elements such as spectrin and dystrophin, and iv) intracellular cytoskeletal proteins such as vimentin, vincu- lin, synemin, desmin and actin, as well as v) various tubu- lins that form microtubules. Differential effects of muscle aging have previously been observed to occur in extra-sar- comeric proteins.60-65,79-86 Increases in the major intermediate filament protein desmin indicate a certain degree of remod- eling of cytoskeletal networks in senescent myofibers. This could be a compensatory mechanism to stabilize the weak- ened intracellular structures of aged muscle fibers. Proteomic profiling of extracellular matrix proteins in aged skeletal muscle In skeletal muscles, the extracellular matrix is organized into three complex layers that form the endomysium, peri- mysium and epimysium, which are intrinsically involved in the provision of force transmission, as well as structural support, maintenance of the microenvironment of diverse myofiber populations and repair mechanisms. As listed in Table 3, the mass spectrometric analysis of aged mouse hindlimb muscles identified key components that are mostly found in the basal lamina (collagens COL-IV and COL-XV, laminin-211, perlecan, and the nidogen isoforms entactin and osteonidogen) or the endomysium (collagens COL-V and COL-VI). Proteins that are present throughout the endomysium, perimysium and epimysium were recog- - 72 - Table 1. Continued from previous page. Accession Protein name Gene Peptides Coverage Molecular mass (%) (kDa) (iv) Thin filament actin complex and troponin/tropomyosin regulatory complex P68033 Actin, alpha, cardiac muscle Actc1 32 82.2 42.0 P68134 Actin, alpha, skeletal muscle Acta1 32 81.2 42.1 P20801 Troponin TnC, muscle Tnnc2 11 78.1 18.1 P19123 Troponin TnC, slow muscle Tnnc1 6 41.0 18.4 O88346 Troponin TnT, slow muscle Tnnt1 7 24.8 31.3 Q9QZ47 Troponin TnT, fast muscle Tnnt3 17 36.8 32.2 Q9WUZ5 Troponin TnI, slow muscle Tnni1 10 44.4 21.7 P13412 Troponin TnI, fast muscle Tnni2 10 42.3 21.4 P58771 Tropomyosin, alpha-1 Tpm1 37 78.2 32.7 P21107 Tropomyosin, alpha-3 Tpm3 33 75.4 33.0 Q6IRU2 Tropomyosin, alpha-4 Tpm4 15 58.9 28.5 P47753 F-actin-capping protein, alpha-1 Capza1 11 59.4 32.9 P47754 F-actin-capping protein, alpha-2 Capza2 16 74.1 33.0 P47757 F-actin-capping protein, beta Capzb 20 66.1 31.4 P49813 Tropomodulin-1 Tmod1 14 56.5 40.5 Q9JHJ0 Tropomodulin-3 Tmod3 7 22.2 39.5 Q9JLH8 Tropomodulin-4 Tmod4 10 40.3 39.3 O70373 Xin actin-binding protein 1 Xirp1 8 11.8 123.4 Q5DTJ9 Myopalladin Mypn 8 10.2 144.1Non -co mmerc ial us e o nly Proteomic reference map for sarcopenia research Part 1 Eur J Transl Myol 34 (2) 12564, 2024 doi: 10.4081/ejtm.2024.12564 nized as dermatopontin, vitronectin, fibronectin, fibri- nogens, prolargin and fibrillin (Table 3). Members of the large family of small leucine-rich pro- teoglycans (SLRP) were clearly identified in aged mus- cles, including decorin, asporin, biglycan, fibromodulin and mimecan/osteoglycin. Additional protein species that are linked to the extracellular matrix are listed in Table 3, i.e. the sarcolemmal adhesion proteins/linkers alpha/beta- dystroglycan and alpha7/beta1-integrin, as well as the an- nexins ANXA2 and ANXA6. Markers of the myotendinous junction, tendon and cartilage are listed in the form of collagen COL-XII, collagen COL-I and carti- lage-associated protein, respectively. Previous studies of aged muscle do not suggest a major increase in matriso- mal proteins, such as collagens and proteoglycans,60-65,79- 86 so sarcopenia of old age is probably associated with a lower degree of myofibrosis as compared to severe neu- romuscular disorders, such as X-linked muscular dystro- phy.56 This has been confirmed in this study, as can be seen in the below immunoblot analysis of the abundance of col- lagen isoform COL-VI in aged wild type versus aged and dystrophic mdx-4cv skeletal muscle preparations. - 73 - Table 2. Mass spectrometry-based proteomic profiling of extra-sarcomeric proteins in aged mouse hindlimb muscle. Accession Protein name Gene Peptides Coverage Molecular mass (%) (kDa) Q9QXS1 Plectin (Plec-1d) Plec 283 60.8 534.2 P16546 Spectrin a, non-erythrocytic 1 Sptan1 104 49.3 284.6 Q62261 Spectrin b, non-erythrocytic 1 Sptbn1 90 49.6 274.2 P20152 Vimentin Vim 64 88.8 53.7 Q64727 Vinculin Vcl 57 67.3 116.7 Q70IV5 Synemin Synm 24 21.1 173.2 P31001 Desmin Des 31 67.8 53.5 P60710 Actin, cytoplasmic 1 Actb 36 95.5 41.7 P63260 Actin, cytoplasmic 2 Actg1 36 95.5 41.8 P11531 Dystrophin (Dp427-M) Dmd 5 1.6 425.8 P05213 Tubulin alpha-1B Tuba1b 34 74.9 50.2 P68373 Tubulin alpha-1C Tuba1c 34 75.1 49.9 P05214 Tubulin alpha-3 Tuba3a 23 51.3 50.0 P68368 Tubulin alpha-4A Tuba4a 27 61.2 49.9 Q9JJZ2 Tubulin alpha-8 Tuba8 20 49.9 50.1 Q7TMM9 Tubulin beta-2A Tubb2a 21 65.6 49.9 Q9CWF2 Tubulin beta-2B Tubb2b 22 75.3 50.0 Q9ERD7 Tubulin beta-3 Tubb3 14 34.4 50.4 Q9D6F9 Tubulin beta-4A Tubb4a 21 69.1 49.6 P99024 Tubulin beta-5 Tubb5 27 80.6 49.7 Q922F4 Tubulin beta-6 Tubb6 18 57.9 50.1 Q8C8R3 Ankyrin-2 Ank2 3 1.1 426.3 G5E8K5 Ankyrin-3 Ank3 5 4.1 214.1 Non -co mmerc ial us e o nly Proteomic reference map for sarcopenia research Part 1 Eur J Transl Myol 34 (2) 12564, 2024 doi: 10.4081/ejtm.2024.12564 - 74 - Table 3. Mass spectrometry-based proteomic profiling of matrisomal proteins in aged mouse hindlimb muscle. Accession Protein name Gene Peptides Coverage Molecular mass (%) (kDa) (i) Basal lamina P02463 Collagen IV, alpha-1 Col4a1 3 2.3 160.7 P08122 Collagen IV, alpha-2 Col4a2 7 6.9 167.3 O35206 Collagen XV, alpha-1 Col15a1 7 8.3 140.5 Q60675 Laminin-211, subunit alpha-2 Lama2 64 31.0 343.8 P02469 Laminin-211, subunit beta-1 Lamb1 36 24.9 197.1 P02468 Laminin-211, subunit gamma-1 Lamc1 52 42.8 177.3 Q05793 Perlecan (HSPG-2) Hspg2 53 21.6 398.3 P10493 Nidogen-1 (entactin) Nid1 23 24.3 136.54 O88322 Nidogen-2 (osteonidogen) Nid2 8 7.8 153.91 (ii) Endomysium Q3U962 Collagen V, alpha-2 Col5a2 4 4.1 145.0 Q04857 Collagen VI, alpha-1 Col6a1 27 43.0 108.5 Q02788 Collagen VI, alpha-2 Col6a2 22 26.3 110.3 A6H584 Collagen VI, alpha-5 Col6a5 5 2.2 289.6 Q8C6K9 Collagen VI, alpha-6 Col6a6 8 5.2 246.3 (iii) Endomysium, perimysium and epimysium Q9QZZ6 Dermatopontin Dpt 6 33.8 24.0 P29788 Vitronectin Vtn 3 14.2 54.9 P11276 Fibronectin Fn1 22 13.4 272.5 E9PV24 Fibrinogen, alpha Fga 13 21.7 87.4 Q8K0E8 Fibrinogen, beta Fgb 16 50.5 54.8 Q8VCM7 Fibrinogen, gamma Fgg 14 33.7 49.4 Q9JK53 Prolargin Prelp 14 40.7 43.3 Q61554 Fibrillin-1 Fbn1 59 28.9 312.3 (iv) Small leucine-rich proteoglycans (SLRP type) P28654 Decorin Dcn 15 46.6 39.8 Q99MQ4 Asporin Aspn 11 33.5 42.6 P28653 Biglycan Bgn 11 38.2 41.6 P50608 Fibromodulin Fmod 4 18.4 43.1 Q62000 Mimecan (Osteoglycin) Ogn 11 42.6 34.0 To be continued on next page Non -co mmerc ial us e o nly Proteomic reference map for sarcopenia research Part 1 Eur J Transl Myol 34 (2) 12564, 2024 doi: 10.4081/ejtm.2024.12564 Bioinformatic analysis of potential protein-protein interactions within protein clusters in aged skeletal muscle The various muscle-associated protein complexes that were identified by mass spectrometry-based proteomics were further analyzed in the context of potential protein interaction patterns. As outlined in Figure 3, bioinfor- matic STRING analyses,77 clearly demonstrated consid- erable interaction patterns within protein clusters that are involved in the contraction-relaxation cycle, the extra- sarcomeric cytoskeleton and the complex arrangement of the matrisome in the extracellular matrix. Due to the com- plexity of functional and physical protein clustering and the large number of interacting protein species, the illus- trations depicted in Figure 3 are not presented to give de- tailed information on individual protein interactions, but instead are shown to provide a general overview of com- plex formation of identified protein groupings in aged skeletal muscles. Identification of proteomic markers of dystrophinopathy in aged mdx-4cv muscle To demonstrate the usefulness of the newly established reference map of aged skeletal muscle, the comparison of wild type muscle versus dystrophic muscle was carried out using the established murine mdx-4cv model of Duch- enne muscular dystrophy.39 The marker proteins listed in Table 4 are molecular species that are absent versus pres- ent in the two different muscle specimens as judged by mass spectrometric surveys, analyzed using identical chromatography and protein identification parameters. These types of comparisons are ideal for single tissue sam- ple classification purposes. In the case of the dystrophic samples, fibrotic markers such as the matricellular protein periostin and collagen isoform COL-XVIII, are present, in addition to moesin of the ezrin-radixin-moesin protein family that mediates linking of the plasmalemma to the actin cytoskeleton, and the cysteine protease cathepsin-B that is involved in intracellular proteolysis. In contrast, dystrophin isoform Dp427-M, which is primarily affected in X-linked muscular dystrophy,68 is only present in wild type muscle, in conjunction with its associated glycopro- teins dystroglycan and sarcoglycans. These clear pro- teomic differences between aged wild type versus aged mdx-4cv mouse hindlimb muscle demonstrate the suitabil- ity of simple data-based searches for tissue classification. The main advantage of this straightforward approach using established proteomic markers is the fact that it is considerably less time consuming as compared to soft- ware-driven analyses. The proteomic characterization of the wild type versus dystrophic specimens confirms that the absence of dystrophin isoform Dp427-M and collapse of the dystro- phin-associated glycoprotein complex are the primary trigger of sarcolemmal damage in this murine model of Duchenne muscular dystrophy. Increased levels of myonecrosis were previously shown to lead to fat substi- tution and chronic inflammation in dystrophic muscles. - 75 - Table 3. Continued from previous page. Accession Protein name Gene Peptides Coverage Molecular mass (%) (kDa) (v) Sarcolemmal adhesion proteins/linkers Q62165 Dystroglycan, alpha/beta Dag1 12 15.5 96.9 Q61738 Integrin, alpha-7 Itga7 8 8.7 129.3 P09055 Integrin, beta-1 Itgb1 23 38.0 88.2 (vi) Myotendinous junction, tendon and cartilage Q60847 Collagen XII, a-1(myotendinous junction) Col12a1 21 8.9 340.2 P11087 Collagen I, alpha-1 (tendon) Col1a1 9 8.0 138.0 Q01149 Collagen I, alpha-2 (tendon) Col1a2 10 10.1 129.6 Q9CYD3 Cartilage-associated protein Crtap 8 18.5 46.2 (vii) Annexins P07356 Annexin A2 Anxa2 41 85.3 38.7 P14824 Annexin A6 Anxa6 47 65.4 75.9 Non -co mmerc ial us e o nly Proteomic reference map for sarcopenia research Part 1 Eur J Transl Myol 34 (2) 12564, 2024 doi: 10.4081/ejtm.2024.12564 - 76 - Figure 3. 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 the contractile apparatus in sarcomeres (Table 1), the extra-sar- comeric cytoskeletal network (Table 2) and matrisomal proteins of the extracellular matrix (Table 3). The publicly available bioinformatic analysis tool STRING (https://string-db.org) was used for the analysis of protein interaction patterns.77 Non -co mmerc ial us e o nly Proteomic reference map for sarcopenia research Part 1 Eur J Transl Myol 34 (2) 12564, 2024 doi: 10.4081/ejtm.2024.12564 Proteomics is an ideal bioanalytical tool to study the com- plexity of neuromuscular changes and multi-systems dis- turbances in dystrophinopathy, as recently discussed in a review on pathophysiological crosstalk in X-linked mus- cular dystrophy.87 Reactive myofibrosis is a characteristic hallmark of Duchenne muscular dystrophy and this was confirmed here by the drastic increase in the matricellular component periostin and a specific isoform of collagen.56 Since the presence of periostin, a protein that is almost ab- sent from normal skeletal muscle, was clearly established in senescent mdx-4cv mouse hindlimb muscle, the dys- trophic phenotype appears to be characterized by high levels of reactive myofibrosis. One of the most frequently employed fibrosis markers in the field of skeletal muscle pathobiology is the COL-VI isoform of collagen. As dis- played in Figure 4, comparative immunoblotting showed an increase in the abundance of this key element of the ex- tracellular matrix in the dystrophic and fibrotic mdx-4cv specimens. In contrast, CBB staining of gels did not show any major differences in protein expression patterns, and immunoblotting with antibodies to glyceraldehyde 3-phos- phate dehydrogenase, tropomyosin and parvalbumin did not reveal changes in their abundance in aged wild type versus aged mdx-4cv mouse hindlimb muscle. Discussion This study has focused on the mass spectrometric profiling of total tissue extracts that were isolated from the aged mouse hindlimb musculature. The major aim of this inves- tigation was to establish a proteomic reference map of se- nescent mouse skeletal muscles using a streamlined, economical and bottom-up proteomic workflow. The newly established proteomic catalogue displayed in this report in- cludes key muscle proteins that are associated with the sar- comere, the extra-sarcomeric cytoskeleton and the extracellular matrix. These listings of unequivocally iden- tified skeletal muscle proteins can now be employed as a biochemical reference for future investigations that will at- tempt to further elucidate the highly complex and multi- factorial mechanisms that underlie sarcopenia of old age.31 The processes that underlie sarcopenic changes are highly complex, including i) an increased rate of muscular atrophy in fast-twitching myofibers causing preferentially fast-to- slow muscle transitions, ii) neurodegeneration, which is characterized by the disintegration of motor neurons result- ing in denervation and faulty patterns of reinnervation, as well as the progressive loss of the functionality of the neu- romuscular junction system, iii) uncoupling between sar- colemmal excitation and contraction at the level of triad junctions, iv) abnormal calcium handling in the cytosol, sar- coplasmic reticulum and mitochondria, v) indirect bioener- getic shifting from glycolytic to more oxidative metabolism in slower contracting muscles, vi) proteotoxic effects trig- gering a sustained cellular stress response, vii) imbalanced proteostasis due to abnormal rates of protein turn-over and re-synthesis, viii) hormonal disturbances, including insulin resistance, and abnormal cellular signaling, affecting ma- jorly the muscle-fat axis ix) a certain degree of myofibrosis that causes negative effects on myofiber elasticity and lat- eral force transmission, x) chronic inflammation, xi) epig- enetic alterations, and xii) stem cell exhaustion that is associated with a reduction in regenerative capacity.20-24,30 - 77 - Table 4. Mass spectrometry-based proteomic profiling of protein markers in aged wild type versus aged mdx-4cv mouse hindlimb muscle. Accession Protein name Gene Peptides Coverage Molecular mass (%) (kDa) (i) Proteins uniquely identified in aged and dystrophic mdx-4cv muscle Q62009 Periostin Postn 8 13 93.1 P39061 Collagen alpha-1(XVIII) Col18a1 2 2 182.1 P26041 Moesin Msn 9 12 67.7 P10605 Cathepsin B Ctsb 5 24 37.3 (ii) Proteins uniquely identified in aged wild type muscle P11531 Dystrophin Dp427-M Dmd 8 4 425.6 Q62165 Dystroglycan Dag1 1 2 96.8 P82349 Sarcoglycan, beta Sgcb 1 7 34.9 P82347 Sarcoglycan, delta Sgcd 1 3 32.1 Non -co mmerc ial us e o nly Proteomic reference map for sarcopenia research Part 1 Eur J Transl Myol 34 (2) 12564, 2024 doi: 10.4081/ejtm.2024.12564 Skeletal muscle proteomics is an unbiased screening ap- proach in discovery mode that can be used in bottom-up, middle-up/down or top-down fashion to identify and char- acterize distinct protein species and their post-translational modifications.32 For the comprehensive protein biochemical analysis of complex muscle tissues, one of the most cost- effective and experimentally straightforward approaches is the label-free liquid chromatography-tandem mass spectro- metric screening of total protein extracts from crude tissue homogenates.31,88,89 Bottom-up proteomics is based on the identification of distinct protein species through the char- acterization of peptides that are produced from proteins of interest by controlled proteolysis.90 This widely used ap- proach ultimately detects and measures peptides, which rep- resent ideal molecular species for the swift proteomic analysis due to the fact that they readily solubilize, separate and ionize.91-93 Column chromatography is routinely em- ployed for efficient protein separation, utilizing differences in charge (ion exchange – anion/cation), affinity (a specific binding affinity for peptide moieties or post-translational modifications) or size (gel filtration/size exclusion - resins are porous to molecules with a particular size range). A key step is the efficient digestion of proteins into peptides prior to MS-based analysis using either bottom-up proteomics or top-down proteomics. Trypsin is highly suitable for digest- ing proteins into small-size peptides,94 which are more ame- nable to high-performance liquid chromatography separation and tandem mass spectrometric characterization. Alternative enzymes are available to be used alone or in combination with trypsin for the optimum generation of peptide populations prior to mass spectrometric analysis.95- 97 Once peptides have been generated after digestion, silica based octadecyl (C18) resins can be employed to purify and concentrate established peptides. In bottom-up approaches, sophisticated liquid chromatog- raphy is instrumental for the optimum separation of pep- tides prior to mass spectrometry. Of note, the use of first dimension methods (e.g. size exclusion chromatography/ SEC or ion exchange chromatography/IEX) coupled to compatible second dimension approaches (e.g. reversed- phase liquid chromatography/RPLC or hydrophilic inter- action liquid chromatography/HILIC) and related techniques, greatly improves the resolving power and thereby decisively increases the number of peptides that can be analyzed per run.98 The usage of untargeted label- free quantitation (LFQ) of protein species enables the de- termination of the relative abundance of peptides/proteins using both spectral counting and measuring MS1 signal intensities.99-101 However, ion intensities are more accu- rate than spectral counts and have a greater dynamic range. A potential drawback of the LFQ approach is that run parameters, e.g. C18 column conditions, may change marginally between samples, having occasionally knock- on consequences with respect to sample alignment and analysis. In contrast to a peptide-centric analysis of complex protein mixtures, as described here, an alternative strategy is rep- resented by top-down proteomics, which has been widely - 78 - Figure 4. Comparative immunoblot analysis of aged wild type versus aged mdx-4cv mouse hindlimb muscle. Shown is a CBB stained gel plus identical immunoblots labelled with antibodies to the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH), the sarcomeric protein tropomyosin TPM1-alpha (TPM1) of the actin-containing thin filaments, the cytosolic calcium-binding protein parvalbumin (PVA) and the extracellular matrix protein collagen VI (COL-VI). Lanes 1 and 2 contain protein extracts from 24 months old wild type versus mdx-4cv hindlimb muscles, respectively. A graphical representation of the immuno-decoration levels of collagen COL-VI are shown: Student’s t-test, unpaired; n=4; **p <0.01. Molecular weight standards are marked on the left. Non -co mmerc ial us e o nly Proteomic reference map for sarcopenia research Part 1 Eur J Transl Myol 34 (2) 12564, 2024 doi: 10.4081/ejtm.2024.12564 used in skeletal muscle proteomics in the past,102-104 includ- ing the large-scale analysis of aged myofibers.79-86 Protein separation in top-down proteomics is often gel-based, i.e. two-dimensional gel electrophoresis is used prior to the di- gestion of intact proteoforms.33 High-resolution gels can be used to facilitate the separation of hundreds to thousands of proteins on one 2D-gel. Of note, this technique has been optimized to separate the skeletal muscle proteome.104,105 The advantage of gel-based top-down proteomics is the fact that this workflow can be effectively combined with other biochemical techniques, facilitating gel staining, followed by spot excision, de-staining, protein digestion, and the sub- sequent analysis of peptides by mass spectrometric analysis to unequivocally identify specific proteoforms. However, a major analytical limitation of two-dimensional gel electrophoresis is the lack of sufficient electrophoretic mobility of very large proteins, which hinders the efficient movement of high-molecular-mass muscle protein species, such as dystrophin, plectin, obscurin, the ryanodine receptor calcium release channel, nebulin and titin, from the first di- mension isoelectric focusing tube gels into the matrix of second dimension slab gels. In addition, accurate identifi- cation of proteins of very low abundance and/or species with extreme isoelectric points is not achievable using this technique. Gel-based techniques are considerably more time-consuming, costly and labor-intensive as compared to streamlined bottom-up proteomics. Importantly, since ex- tensive subcellular fractionation steps can introduce con- siderable bioanalytical artefacts, especially in comparative studies,106-109 it is advantageous to use crude extracts for ini- tial studies. Thus, when starting material is scarce, bottom- up proteomics using total protein extracts is the method of choice for comprehensive biochemical surveys of skeletal muscle specimens. The application of bottom-up proteomics in this study has identified the main protein components of the sarcomeres that are involved in the regulation of the contraction-relax- ation cycle and force generation.110-112 This includes a large array of fast and slow isoforms of myosin light and heavy chains in the thick filaments, which are characteristic for the presence of fast- versus slow-twitching myofibers.43-45,113 In analogy, fast versus slow troponin isoforms were identi- fied in the actin-containing thin filaments.113,114 This shows that the presented analysis pipeline is suitable for studying myofiber type shifting during aging.30 Crucial extra-sar- comeric cytoskeletal proteins were identified in skeletal muscles, including the central cytolinker plectin,115 and members of costameres such as alpha7/beta1-integrin,116 and alpha/beta-dystroglycan in conjunction with its mem- brane cytoskeletal anchor named dystrophin.117,118 This would enable the in-depth analysis of alterations in the levels of lateral force transmission through costameric structures in aged skeletal muscles. Since the extracellular matrix is a crucial structural component of muscles,119 and majorly involved in reactive myofibrosis in neuromuscular diseases,120,121 it is encouraging that representative markers of the basal lamina, endomysium, perimysium, epimysium and tendon could be detected by proteomics in crude mus- cle tissue extracts. In conclusion, the mass spectrometric screening of se- nescent mouse muscles has been successfully applied to establish a proteomic reference map for future sarcope- nia research. As shown in this report, proteomic markers of dystrophic and fibrotic changes can easily be identi- fied by simple data-based searches for tissue classifica- tion. For example, the lack of the membrane cytoskeletal protein dystrophin,117 in conjunction with the increase in the matricellular protein periostin,122 is a suitable way of confirming the dystrophic and fibrotic status of a par- ticular muscle sample, such as aged mdx-4cv skeletal muscle tissues.121 List of abbreviations ACT, Actin CBB, Coomassie Brilliant Blue COL, Collagen Dp427-M, Dystrophin of 427 kDa ECL, Enhanced chemiluminescence FASP, Filter-aided sample preparation FLNC, Filamin C GAPDH, Glyceraldehyde 3-phosphate dehydrogenase LFQ, Label-free quantitation MARP, Muscle ankyrin repeat protein MLC, Myosin light chain MS, Mass spectrometry MyBP, Myosin-binding protein MyHC, Myosin heavy chain OSF, Open Science Foundation PVA, Parvalbumin Tn, Troponin TPM, Tropomyosin 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 biochemical experi- ments and analyzed the data. MH and PM performed the mass spectrometric and bioinformatic analysis. All authors were involved in the writing and final editing of the manu- script. 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- - 79 - Non -co mmerc ial us e o nly Proteomic reference map for sarcopenia research Part 1 Eur J Transl Myol 34 (2) 12564, 2024 doi: 10.4081/ejtm.2024.12564 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. Hatton IA, Galbraith ED, Merleau NSC, et al. The human cell count and size distribution. Proc Natl Acad Sci U S A 2023;120:e2303077120. 2. Frontera WR, Ochala J. Skeletal muscle: a brief re- view of structure and function. Calcif Tissue Int 2015;96: 183-95. 3. Mukund K, Subramaniam S. Skeletal muscle: A re- view of molecular structure and function, in health and disease. Wiley Interdiscip Rev Syst Biol Med 2020;12: e1462. 4. Brooks SV, Guzman SD, Ruiz LP. Skeletal muscle structure, physiology, and function. 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