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• W2766450105 abstract "Sarcopenia, the age-related loss of skeletal muscle mass and strength, is a significant cause of morbidity in the elderly and is a major burden on health care systems. Unfortunately, the underlying molecular mechanisms in sarcopenia remain poorly understood. Herein, we utilized top-down proteomics to elucidate sarcopenia-related changes in the fast- and slow-twitch skeletal muscles of aging rats with a focus on the sarcomeric proteome, which includes both myofilament and Z-disc proteins—the proteins that constitute the contractile apparatuses. Top-down quantitative proteomics identified significant changes in the post-translational modifications (PTMs) of critical myofilament proteins in the fast-twitch skeletal muscles of aging rats, in accordance with the vulnerability of fast-twitch muscles to sarcopenia. Surprisingly, age-related alterations in the phosphorylation of Cypher isoforms, proteins that localize to the Z-discs in striated muscles, were also noted in the fast-twitch skeletal muscle of aging rats. This represents the first report of changes in the phosphorylation of Z-disc proteins in skeletal muscle during aging. In addition, increased glutathionylation of slow skeletal troponin I, a novel modification that may help protect against oxidative damage, was observed in slow-twitch skeletal muscles. Furthermore, we have identified and characterized novel muscle type-specific proteoforms of myofilament proteins and Z-disc proteins, including a novel isoform of the Z-disc protein Enigma. The finding that the phosphorylation of Z-disc proteins is altered in response to aging in the fast-twitch skeletal muscles of aging rats opens new avenues for the investigation of the role of Z-discs in age-related muscle dysfunction. Sarcopenia, the age-related loss of skeletal muscle mass and strength, is a significant cause of morbidity in the elderly and is a major burden on health care systems. Unfortunately, the underlying molecular mechanisms in sarcopenia remain poorly understood. Herein, we utilized top-down proteomics to elucidate sarcopenia-related changes in the fast- and slow-twitch skeletal muscles of aging rats with a focus on the sarcomeric proteome, which includes both myofilament and Z-disc proteins—the proteins that constitute the contractile apparatuses. Top-down quantitative proteomics identified significant changes in the post-translational modifications (PTMs) of critical myofilament proteins in the fast-twitch skeletal muscles of aging rats, in accordance with the vulnerability of fast-twitch muscles to sarcopenia. Surprisingly, age-related alterations in the phosphorylation of Cypher isoforms, proteins that localize to the Z-discs in striated muscles, were also noted in the fast-twitch skeletal muscle of aging rats. This represents the first report of changes in the phosphorylation of Z-disc proteins in skeletal muscle during aging. In addition, increased glutathionylation of slow skeletal troponin I, a novel modification that may help protect against oxidative damage, was observed in slow-twitch skeletal muscles. Furthermore, we have identified and characterized novel muscle type-specific proteoforms of myofilament proteins and Z-disc proteins, including a novel isoform of the Z-disc protein Enigma. The finding that the phosphorylation of Z-disc proteins is altered in response to aging in the fast-twitch skeletal muscles of aging rats opens new avenues for the investigation of the role of Z-discs in age-related muscle dysfunction. Sarcopenia, the loss of skeletal muscle mass and function (i.e. strength) with aging, is a significant cause of morbidity and disability in the elderly population (1.Cohen S. Nathan J.A. Goldberg A.L. Muscle wasting in disease: molecular mechanisms and promising therapies.Nat. Rev. Drug Discov. 2015; 14: 58-74Crossref PubMed Scopus (639) Google Scholar, 2.Ryall J.G. Schertzer J.D. Lynch G.S. Cellular and molecular mechanisms underlying age-related skeletal muscle wasting and weakness.Biogerontology. 2008; 9: 213-228Crossref PubMed Scopus (276) Google Scholar, 3.Rolland Y. Czerwinski S. Abellan Van Kan G. Morley J.E. Cesari M. Onder G. Woo J. Baumgartner R. Pillard F. Boirie Y. Chumlea W.M. Vellas B. Sarcopenia: its assessment, etiology, pathogenesis, consequences and future perspectives.J. Nutr. Health Aging. 2008; 12: 433-450Crossref PubMed Scopus (659) Google Scholar). Greater than 35% of persons over 65 years of age and 50% or more of individuals 80 years or older are estimated to be afflicted with sarcopenia (4.Janssen I. Heymsfield S.B. Ross R. Low relative skeletal muscle mass (sarcopenia) in older persons is associated with functional impairment and physical disability.J. Am. Geriatr. Soc. 2002; 50: 889-896Crossref PubMed Scopus (2157) Google Scholar). Moreover, the sarcopenia-associated burden on healthcare is substantial with an estimated cost of $18.5 billion in the United States in the year 2000 alone (5.Janssen I. Shepard D.S. Katzmarzyk P.T. Roubenoff R. The healthcare costs of sarcopenia in the United States.J. Am. Geriatr. Soc. 2004; 52: 80-85Crossref PubMed Scopus (993) Google Scholar); and costs are expected to rise because of aging of the population (6.Wiener J.M. Tilly J. Population ageing in the United States of America: implications for public programmes.Int. J. Epidemiol. 2002; 31: 776-781Crossref PubMed Scopus (194) Google Scholar). Nevertheless, despite the high prevalence and devastating economic impact, among types of systemic muscle loss and weakness, sarcopenia represents one of the least well understood (1.Cohen S. Nathan J.A. Goldberg A.L. Muscle wasting in disease: molecular mechanisms and promising therapies.Nat. Rev. Drug Discov. 2015; 14: 58-74Crossref PubMed Scopus (639) Google Scholar). Although muscle mass declines with age, largely because of the atrophy and loss of type II (fast-twitch) skeletal muscle fibers whereas type I (slow-twitch) fibers have traditionally been thought to remain relatively unaffected (7.Ciciliot S. Rossi A.C. Dyar K.A. Blaauw B. Schiaffino S. Muscle type and fiber type specificity in muscle wasting.Int. J. Biochem. Cell Biol. 2013; 45: 2191-2199Crossref PubMed Scopus (323) Google Scholar, 8.Thompson L.V. Effects of age and training on skeletal muscle physiology and performance.Phys. Ther. 1994; 74: 71-81Crossref PubMed Scopus (109) Google Scholar), the loss of muscle strength in elderly individuals cannot be entirely explained by the age-associated loss of muscle mass (9.Thompson L.V. Age-related muscle dysfunction.Exp. Gerontol. 2009; 44: 106-111Crossref PubMed Scopus (178) Google Scholar). Moreover, even though metabolic perturbations and cell damage resulting from age-related mitochondrial dysfunction undoubtedly contribute to muscle dysfunction with increasing age (10.Johnson M.L. Robinson M.M. Nair K.S. Skeletal muscle aging and the mitochondrion.Trends Endocrinol. Metab. 2013; 24: 247-256Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar), it is now well-recognized that sarcopenia is associated with a loss of intrinsic contractile function that is due, in part, to alterations in myofilaments (11.Miller M.S. Callahan D.M. Toth M.J. Skeletal muscle myofilament adaptations to aging, disease, and disuse and their effects on whole muscle performance in older adult humans.Front. Physiol. 2014; 5: 369Crossref PubMed Scopus (53) Google Scholar). Myofilaments are components of sarcomeres, the basic contractile units of muscle; and are composed of the thin filament proteins actin, tropomyosin (Tpm) 1The abbreviations used are: Tpm, tropomyosin; TnI, troponin I; TnT, troponin T; TnC, troponin C; MHC, myosin heavy chain; PTMs, post-translational modifications; MS, mass spectrometry; MS/MS, tandem MS; ECD, electron capture dissociation; F344BN, Fisher 344 × Brown Norway F1 hybrid; GAS, gastrocnemius; SOL, soleus; CSA, cross-sectional area; LC, liquid chromatography; q-TOF, quadrupole-time-of-flight; LTQ, linear ion trap; FT-ICR, Fourier transform ion cyclotron resonance; Ptotal, total protein phosphorylation; fsTnT, fast skeletal troponin T; fsTnI, fast skeletal troponin I; αTpm, α-tropomyosin; MLC-1F, fast skeletal myosin essential light chain 1; MLC-3F, fast skeletal myosin essential light chain 3; fsTnC, fast skeletal troponin C; MLC-2F, fast skeletal isoform of the myosin regulatory light chain; nEnigma, novel isoform of the Enigma protein; ssTnT1, slow skeletal troponin T isoform 1; ssTnI, slow skeletal troponin I; MLC-1S, slow skeletal myosin essential light chain; ssTnC, slow skeletal troponin C; MLC-1V, slow skeletal/ventricular myosin essential light chain; MLC-2S, slow skeletal/ventricular myosin regulatory light chain; βTpm, β-tropomyosin; pfsTnI, phosphorylated fsTnI; GSS-fsTnI, glutathionylated fsTnI; pαTpm, phosphorylated αTpm; pβTpm, phosphorylated βTpm; pMLC-2F, mono-phosphorylated MLC-2F; ppMLC-2F, bis-phosphorylated MLC-2F; pssTnT1, mono-phosphorylated ssTnT1; pssTnI, mono-phosphorylated ssTnI; GSS-ssTnI, glutathionylated ssTnI; pCypher2s, mono-phosphorylated Cypher2s; pCypher4s, mono-phosphorylated Cypher4s. 1The abbreviations used are: Tpm, tropomyosin; TnI, troponin I; TnT, troponin T; TnC, troponin C; MHC, myosin heavy chain; PTMs, post-translational modifications; MS, mass spectrometry; MS/MS, tandem MS; ECD, electron capture dissociation; F344BN, Fisher 344 × Brown Norway F1 hybrid; GAS, gastrocnemius; SOL, soleus; CSA, cross-sectional area; LC, liquid chromatography; q-TOF, quadrupole-time-of-flight; LTQ, linear ion trap; FT-ICR, Fourier transform ion cyclotron resonance; Ptotal, total protein phosphorylation; fsTnT, fast skeletal troponin T; fsTnI, fast skeletal troponin I; αTpm, α-tropomyosin; MLC-1F, fast skeletal myosin essential light chain 1; MLC-3F, fast skeletal myosin essential light chain 3; fsTnC, fast skeletal troponin C; MLC-2F, fast skeletal isoform of the myosin regulatory light chain; nEnigma, novel isoform of the Enigma protein; ssTnT1, slow skeletal troponin T isoform 1; ssTnI, slow skeletal troponin I; MLC-1S, slow skeletal myosin essential light chain; ssTnC, slow skeletal troponin C; MLC-1V, slow skeletal/ventricular myosin essential light chain; MLC-2S, slow skeletal/ventricular myosin regulatory light chain; βTpm, β-tropomyosin; pfsTnI, phosphorylated fsTnI; GSS-fsTnI, glutathionylated fsTnI; pαTpm, phosphorylated αTpm; pβTpm, phosphorylated βTpm; pMLC-2F, mono-phosphorylated MLC-2F; ppMLC-2F, bis-phosphorylated MLC-2F; pssTnT1, mono-phosphorylated ssTnT1; pssTnI, mono-phosphorylated ssTnI; GSS-ssTnI, glutathionylated ssTnI; pCypher2s, mono-phosphorylated Cypher2s; pCypher4s, mono-phosphorylated Cypher4s., and the troponin complex, which includes troponin I (TnI), troponin T (TnT), and troponin C (TnC), as well as the thick filament proteins myosin heavy chain (MHC), regulatory light chain, essential light chain, and myosin binding protein C. The myofilaments are flanked on either side by protein dense structures known as Z-discs that, together with the myofilaments constitute sarcomeres, which are responsible for mediating muscle contraction at high levels of intracellular Ca2+ (12.Moss R.L. Diffee G.M. Greaser M.L. Contractile properties of skeletal muscle fibers in relation to myofibrillar protein isoforms.Rev. Physiol. Biochem. Pharmacol. 1995; 126: 1-63Crossref PubMed Google Scholar, 13.Schiaffino S. Reggiani C. Molecular diversity of myofibrillar proteins: gene regulation and functional significance.Physiol. Rev. 1996; 76: 371-423Crossref PubMed Scopus (1268) Google Scholar). In addition to regulation by Ca2+, which serves as the ultimate trigger for muscle contraction, myofilament protein-protein interactions (and consequently contractile function) are also regulated via the post-translational modification (PTM) of myofilaments, as well as changes in the expression of contractile protein isoforms (12.Moss R.L. Diffee G.M. Greaser M.L. Contractile properties of skeletal muscle fibers in relation to myofibrillar protein isoforms.Rev. Physiol. Biochem. Pharmacol. 1995; 126: 1-63Crossref PubMed Google Scholar, 13.Schiaffino S. Reggiani C. Molecular diversity of myofibrillar proteins: gene regulation and functional significance.Physiol. Rev. 1996; 76: 371-423Crossref PubMed Scopus (1268) Google Scholar). Evidence in the literature suggests that the oxidative modification of myofilaments may underlie altered contractile function in the skeletal muscles of aging individuals (14.Callahan D.M. Miller M.S. Sweeny A.P. Tourville T.W. Slauterbeck J.R. Savage P.D. Maugan D.W. Ades P.A. Beynnon B.D. Toth M.J. Muscle disuse alters skeletal muscle contractile function at the molecular and cellular levels in older adult humans in a sex-specific manner.J. Physiol. 2014; 592: 4555-4573Crossref PubMed Scopus (44) Google Scholar). Specifically, the PTM of reactive cysteines in MHC has been shown to increase with age and correlates with decreased actin-activated myosin ATPase activity in aged rat skeletal muscles (15.Prochniewicz E. Thomas D.D. Thompson L.V. Age-related decline in actomyosin function.J. Gerontol. A Biol. Sci. Med. Sci. 2005; 60: 425-431Crossref PubMed Scopus (44) Google Scholar). In agreement with findings in human skeletal muscle (16.Miller M.S. Bedrin N.G. Callahan D.M. Previs M.J. Jennings M.E. Ades P.A. Maughan D.W. Palmer B.M. Toth M.J. Age-related slowing of myosin actin cross-bridge kinetics is sex specific and predicts decrements in whole skeletal muscle performance in humans.J. Appl. Physiol. 2013; 115: 1004-1014Crossref PubMed Scopus (80) Google Scholar), we recently identified an age-related decrease in the phosphorylation of RLC in fast-twitch skeletal muscles of aging rats that can account for sarcopenic muscle functional impairments (17.Gregorich Z.R. Peng Y. Cai W. Jin Y. Wei L. Chen A.J. McKiernan S.H. Aiken J.M. Moss R.L. Diffee G.M. Ge Y. Top-Down Targeted Proteomics Reveals Decrease in Myosin Regulatory Light-Chain Phosphorylation That Contributes to Sarcopenic Muscle Dysfunction.J. Proteome Res. 2016; 15: 2706-2716Crossref PubMed Scopus (31) Google Scholar). However, a more comprehensive assessment of changes in other contractile proteins in the fast-twitch skeletal muscles of aging individuals remains lacking. Moreover, emerging evidence indicates that slow skeletal muscles may also be vulnerable to sarcopenia at advanced age (18.Purves-Smith F.M. Sgarioto N. Hepple R.T. Fiber typing in aging muscle.Exerc. Sport. Sci. Rev. 2014; 42: 45-52Crossref PubMed Scopus (75) Google Scholar) and, thus, the examination of changes in contractile protein PTMs during the aging process in these muscles, which may contribute to age-related alterations in contractile function, will be imperative for understanding how sarcopenia impacts muscle function in slow-twitch skeletal muscles. Top-down mass spectrometry (MS)-based proteomics, in which intact proteins are analyzed rather than peptides as in bottom-up proteomics, has supplanted traditional methods of protein analysis as the most powerful method for the comprehensive characterization of proteoforms (a term encompassing the myriad protein forms arising from a single gene, including post-translationally modified forms and those with sequence alterations because of mutations/polymorphisms and/or alternative splicing) (19.Smith L.M. Kelleher N.L. Proteomics C. f. T. D. Proteoform: a single term describing protein complexity.Nat. Methods. 2013; 10: 186-187Crossref PubMed Scopus (884) Google Scholar). The analysis of intact proteoforms in top-down proteomics provides a “bird's eye” view of all protein sequence variations and PTMs, which can subsequently be localized using a variety of tandem MS (MS/MS) techniques, such as collision induced dissociation and electron capture dissociation (ECD) (20.Siuti N. Kelleher N.L. Decoding protein modifications using top-down mass spectrometry.Nat. Methods. 2007; 4: 817-821Crossref PubMed Scopus (387) Google Scholar, 21.Gregorich Z.R. Ge Y. Top-down proteomics in health and disease: challenges and opportunities.Proteomics. 2014; 14: 1195-1210Crossref PubMed Scopus (138) Google Scholar, 22.Zhang H. Ge Y. Comprehensive analysis of protein modifications by top-down mass spectrometry.Circ. Cardiovasc. Genet. 2011; 4: 711Crossref PubMed Scopus (109) Google Scholar). Top-down MS in combination with ECD, in particular, is useful for the localization of labile PTMs (e.g. glycosylation and phosphorylation) (23.Zubarev R.A. Horn D.M. Fridriksson E.K. Kelleher N.L. Kruger N.A. Lewis M.A. Carpenter B.K. McLafferty F.W. Electron capture dissociation for structural characterization of multiply charged protein cations.Anal. Chem. 2000; 72: 563-573Crossref PubMed Scopus (850) Google Scholar, 24.Zhang J. Zhang H. Ayaz-Guner S. Chen Y.C. Dong X. Xu Q. Ge Y. Phosphorylation, but not alternative splicing or proteolytic degradation, is conserved in human and mouse cardiac troponin T.Biochemistry. 2011; 50: 6081-6092Crossref PubMed Scopus (29) Google Scholar, 25.Dong X. Sumandea C.A. Chen Y.C. Garcia-Cazarin M.L. Zhang J. Balke C.W. Sumandea M.P. Ge Y. Augmented phosphorylation of cardiac troponin I in hypertensive heart failure.J. Biol. Chem. 2012; 287: 848-857Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 26.Peng Y. Gregorich Z.R. Valeja S.G. Zhang H. Cai W. Chen Y.C. Guner H. Chen A.J. Schwahn D.J. Hacker T.A. Liu X. Ge Y. Top-down proteomics reveals concerted reductions in myofilament and Z-disc protein phosphorylation after acute myocardial infarction.Mol. Cell. Proteomics. 2014; 13: 2752-2764Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 27.Zhang J. Guy M.J. Norman H.S. Chen Y.C. Xu Q. Dong X. Guner H. Wang S. Kohmoto T. Young K.H. Moss R.L. Ge Y. Top-down quantitative proteomics identified phosphorylation of cardiac troponin I as a candidate biomarker for chronic heart failure.J. Proteome Res. 2011; 10: 4054-4065Crossref PubMed Scopus (141) Google Scholar, 28.Medzihradszky K.F. Zhang X. Chalkley R.J. Guan S. McFarland M.A. Chalmers M.J. Marshall A.G. Diaz R.L. Allis C.D. Burlingame A.L. Characterization of Tetrahymena histone H2B variants and posttranslational populations by electron capture dissociation (ECD) Fourier transform ion cyclotron mass spectrometry (FT-ICR MS).Mol. Cell. Proteomics. 2004; 3: 872-886Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 29.Ge Y. Rybakova I.N. Xu Q. Moss R.L. Top-down high-resolution mass spectrometry of cardiac myosin binding protein C revealed that truncation alters protein phosphorylation state.Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 12658-12663Crossref PubMed Scopus (130) Google Scholar, 30.Ansong C. Wu S. Meng D. Liu X. Brewer H.M. Deatherage Kaiser B.L. Nakayasu E.S. Cort J.R. Pevzner P. Smith R.D. Heffron F. Adkins J.N. Pasa-Tolic L. Top-down proteomics reveals a unique protein S-thiolation switch in Salmonella Typhimurium in response to infection-like conditions.Proc. Natl. Acad. Sci. U.S.A. 2013; 110: 10153-10158Crossref PubMed Scopus (124) Google Scholar, 31.Pesavento J.J. Mizzen C.A. Kelleher N.L. Quantitative analysis of modified proteins and their positional isomers by tandem mass spectrometry: human histone H4.Anal. Chem. 2006; 78: 4271-4280Crossref PubMed Scopus (201) Google Scholar), which are preferentially lost when proteins are dissociated with slow heating methods such as collision induced dissociation (24.Zhang J. Zhang H. Ayaz-Guner S. Chen Y.C. Dong X. Xu Q. Ge Y. Phosphorylation, but not alternative splicing or proteolytic degradation, is conserved in human and mouse cardiac troponin T.Biochemistry. 2011; 50: 6081-6092Crossref PubMed Scopus (29) Google Scholar). Moreover, given that PTMs such as phosphorylation and minor sequence variations do not significantly impact the ionization efficiency of intact proteins as they do with peptides (32.Steen H. Jebanathirajah J.A. Rush J. Morrice N. Kirschner M.W. Phosphorylation analysis by mass spectrometry: myths, facts, and the consequences for qualitative and quantitative measurements.Mol. Cell. Proteomics. 2006; 5: 172-181Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar), top-down proteomics is inherently semiquantitative with respect to the relative abundances of protein proteoforms observed within the same spectrum (20.Siuti N. Kelleher N.L. Decoding protein modifications using top-down mass spectrometry.Nat. Methods. 2007; 4: 817-821Crossref PubMed Scopus (387) Google Scholar, 21.Gregorich Z.R. Ge Y. Top-down proteomics in health and disease: challenges and opportunities.Proteomics. 2014; 14: 1195-1210Crossref PubMed Scopus (138) Google Scholar, 22.Zhang H. Ge Y. Comprehensive analysis of protein modifications by top-down mass spectrometry.Circ. Cardiovasc. Genet. 2011; 4: 711Crossref PubMed Scopus (109) Google Scholar). Indeed, our group and others have employed top-down MS to identify proteoform alterations toward a better understanding of the molecular mechanisms of disease (25.Dong X. Sumandea C.A. Chen Y.C. Garcia-Cazarin M.L. Zhang J. Balke C.W. Sumandea M.P. Ge Y. Augmented phosphorylation of cardiac troponin I in hypertensive heart failure.J. Biol. Chem. 2012; 287: 848-857Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 26.Peng Y. Gregorich Z.R. Valeja S.G. Zhang H. Cai W. Chen Y.C. Guner H. Chen A.J. Schwahn D.J. Hacker T.A. Liu X. Ge Y. Top-down proteomics reveals concerted reductions in myofilament and Z-disc protein phosphorylation after acute myocardial infarction.Mol. Cell. Proteomics. 2014; 13: 2752-2764Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 27.Zhang J. Guy M.J. Norman H.S. Chen Y.C. Xu Q. Dong X. Guner H. Wang S. Kohmoto T. Young K.H. Moss R.L. Ge Y. Top-down quantitative proteomics identified phosphorylation of cardiac troponin I as a candidate biomarker for chronic heart failure.J. Proteome Res. 2011; 10: 4054-4065Crossref PubMed Scopus (141) Google Scholar, 33.Chamot-Rooke J. Mikaty G. Malosse C. Soyer M. Dumont A. Gault J. Imhaus A.F. Martin P. Trellet M. Clary G. Chafey P. Camoin L. Nilges M. Nassif X. Duménil G. Posttranslational modification of pili upon cell contact triggers N. meningitidis dissemination.Science. 2011; 331: 778-782Crossref PubMed Scopus (143) Google Scholar). In this study, we used quantitative top-down proteomics to gain insights into age-related molecular changes in fast- and slow-twitch skeletal muscles, with a particular emphasis on changes occurring in the sarcomeric proteome. Interestingly, in addition to the identification of the major muscle type-specific isoforms of myofilament proteins, we detected and characterized several Z-disc proteins, including two isoforms of Cypher, one of which has previously only been detected at the mRNA level, as well as a novel isoform of the protein Enigma. Consistent with the well-established susceptibility of fast-twitch muscle fibers to sarcopenia, top-down quantitative proteomics identified significant changes in the PTMs of several important myofilament proteins in the fast-twitch skeletal muscles of aging rats. Interestingly, although we detected few changes in myofilament protein PTMs in the slow-twitch skeletal muscles of aging rats, a significant increase in the glutathionylation of slow skeletal troponin I (ssTnI), a modification that may help protect against oxidative damage and contribute to differences in the presentation of sarcopenia in fast- and slow-twitch skeletal muscles, was noted. Top-down proteomics further enabled the identification of novel phosphorylated proteoforms of fast- and slow-skeletal troponin I. For the first time, we also provide evidence that the phosphorylation of Z-disc proteins is altered with age in fast-twitch skeletal muscles. Male Fisher 344 × Brown Norway F1 hybrid (F344BN) rats aged 6- (n = 12), 24- (n = 12), and 36-months (n = 12) were obtained from the National Institute on Aging colony maintained by Harlan Sprague-Dawley (Indianapolis, IN). Rats were individually housed in clear plastic cages on a 12 h/12 h light/dark cycle with access to food and water ad libitum. Handling and euthanasia were carried out under the guidelines of the University of Wisconsin-Madison Animal Use and Care Committee. Rats were anesthetized by inhalation of isoflurane, and the gastrocnemius (GAS) and soleus (SOL) muscles from the left hind limb were dissected from origin to insertion and immediately weighed. Muscles were bisected at the mid-belly, embedded in optimal cutting temperature compound (Tissue-Tek; Andwin Scientific, Addison, IL), frozen in liquid N2, and stored at −80 °C for later sectioning. Three consecutive sections (10 μm thick) were cut, starting at the mid-belly, placed on labeled Probe-On Plus microscope slides (Fisher Scientific, Pittsburgh, PA), and stored at −80 °C until use. The first section of each series was stained with hematoxylin and eosin. Sections were photographed using an Olympus BH2 microscope (Olympus, Tokyo, Japan) with an Olympus DP70 digital camera and mid-belly composites of each muscle section were reconstructed by interlacing the images using ImagePro Plus software (Media Cybernetics, Atlanta, GA). For fiber counts, individual muscle fibers were annotated on the composite image of the entire muscle cross-section at the mid-belly, using Adobe Photoshop (Adobe Systems, Inc., San Jose, CA), and total count was tabulated. The whole muscle cross-sectional area (CSA) at the mid-belly was measured by tracing an outline of each muscle using ImagePro Plus. To measure individual muscle fiber CSA, four images (10×) from the H&E sections were captured from GAS and SOL muscles from rats in each age group. A grid with 25 random dots was placed over the images, and the CSA of fibers marked with the dots was measured. Six hundred fibers were measured from each muscle at each age ((4 images per muscle) × (25 fibers per image) × (6 animals per age group) = 600 fibers). GAS and SOL muscles were dissected from the right hind limb as described above, but were immediately flash frozen in liquid N2 and stored at −80 °C for subsequent proteomic analysis. For ATPase staining, 10 μm-thick, transverse frozen sections of muscle were cut using a cryostat (−20 °C) and placed on Probe-On Plus slides (Fisher Scientific). Staining for myosin ATPase to determine fiber type distribution was performed according to Hintz et al. (34.Hintz C.S. Coyle E.F. Kaiser K.K. Chi M.M. Lowry O.H. Comparison of muscle fiber typing by quantitative enzyme assays and by myosin ATPase staining.J. Histochem. Cytochem. 1984; 32: 655-660Crossref PubMed Scopus (75) Google Scholar). Fiber type was determined based on myosin ATPase sensitivity to pH and differences in fiber type staining intensities. Preincubation at pH 10.2 distinguishes between type I (light staining) and type II (dark staining) fibers. At acidic pH (4.5 and 4.25), type I fibers will stain dark. Type II fibers can be further classified into IIa (stain light in both pH 4.5 and 4.25), IIb (stain medium at pH 4.5 but light at pH 4.25), and IIc (stain intermediate at both pH 4.5 and 4.25) fibers. The preincubation solutions were: (1.Cohen S. Nathan J.A. Goldberg A.L. Muscle wasting in disease: molecular mechanisms and promising therapies.Nat. Rev. Drug Discov. 2015; 14: 58-74Crossref PubMed Scopus (639) Google Scholar) 20 mm sodium barbital, 18 mm calcium chloride at pH 10.2 for 15 min or (2 and 3) 50 mm sodium acetate, 30 mm sodium barbital at pH 4.5 or 4.25 for 6 min. All sections were then incubated for 30 min at 37 °C in 20 mm sodium barbital, 9 mm calcium chloride, and 2.7 mm ATP at pH 9.4. Sections were subsequently rinsed in 1% calcium chloride (3 × 3 min each), immersed in 2% cobaltum chloride (3 min), rinsed in 10 changes of tap H2O, stained in 1% ammonium sulfide (30 s), washed in tap running H2O (4 min), dehydrated with ethanol, cleared in xylene, and mounted in Permount (Fisher, Fair Lawn, NJ). Protein extraction was carried out as previously described (26.Peng Y. Gregorich Z.R. Valeja S.G. Zhang H. Cai W. Chen Y.C. Guner H. Chen A.J. Schwahn D.J. Hacker T.A. Liu X. Ge Y. Top-down proteomics reveals concerted reductions in myofilament and Z-disc protein phosphorylation after acute myocardial infarction.Mol. Cell. Proteomics. 2014; 13: 2752-2764Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). Briefly, ∼10 mg of tissue from the GAS (n = 6 for each age group) or SOL (n = 6 for each age group) muscles was homogenized in 100 μl of HEPES extraction buffer containing protease and phosphatase inhibitors (25 mm HEPES pH 7.5, 50 mm NaF, 0.25 mm Na3VO4, 0.25 mm PMSF, 2.5 mm EDTA) using a Teflon pestle (1.5 ml tube rounded tip; Scienceware, Pequannock, NJ). The resulting homogenate was centrifuged at 16,000 × g for 15 min at 4 °C, and the supernatant was discarded. The insoluble pellet was then re-homogenized in 100 μl of TFA extraction buffer (1% trifluoroacetic acid, 1 mm tris(2-carboxyethyl)phosphine) to extract a protein sub-proteome enriched in sarcomeric proteins. After centrifugation (16,000 × g, 4 °C, 25 min), the supernatant was collected and used for on-line top-down proteomic analysis. At least two technical (extraction) replicates were performed per biological replicate. Protein extracts prepared from aging rat GAS and SOL muscles were separated using a nanoACQUITY LC system (Waters, Milford, MA) equipped with a home-packed PLRP column (PLRP-S, 200 mm × 500 μm, 10 μm, 1000 Å; Varian, Lake Forest, CA) and a gradient going from 20% B to 90% B (solvent A: 0.10% formic acid in water; solvent B: 0.10% formic acid in a 50:50 mixture of acetonitrile and ethanol) over 40 min at a flow rate of 8 μl/min. The nanoACQUITY LC system was coupled on-line with an impact II quadrupole-time-of-flight (q-TOF) mass spectrometer (Bruker Daltonics, Billerica, MA). Spectra were collected using a scan rate of 1 Hz over a 500–3000 m/z range. Sarcomeric protein separation and the relative ab" @default.
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• W2766450105 title "Novel Sarcopenia-related Alterations in Sarcomeric Protein Post-translational Modifications (PTMs) in Skeletal Muscles Identified by Top-down Proteomics" @default.
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