Protein Digestion for 2D-DIGE Analysis

Murphy, Sandra; Ohlendieck, Kay

doi:10.1007/978-1-0716-2831-7_23

Cited by 6 publications

(4 citation statements)

References 50 publications

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“…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 digesting proteins into small-size peptides, 94 which are more amenable 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.…”

Section: Discussionmentioning

confidence: 99%

Proteomic reference map for sarcopenia research: mass spectrometric identification of key muscle proteins located in the sarcomere, cytoskeleton and the extracellular matrix

Dowling,

Gargan,

Zweyer

et al. 2024

Eur J Transl Myol

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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.

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Section: Discussionmentioning

confidence: 99%

Proteomic reference map for sarcopenia research: mass spectrometric identification of key muscle proteins located in the sarcomere, cytoskeleton and the extracellular matrix

Dowling,

Gargan,

Zweyer

et al. 2024

Eur J Transl Myol

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“…Silver stain and CBB [ 72 ] are robust staining methods facilitating the quick visualization of results [ 73 ], while Pro-Q Emerald and Pro-Q Diamond are specific to glycosylated and phosphorylated proteins, respectively, conferring a degree of specialization with this approach. Trypsin is the gold standard for protein digestion [ 81 ]; however, alternative proteases, such as endoproteinase Glu-C, endoproteinase Asp-N or chymotrypsin, can help increase amino acid sequence coverage by generating unique peptides that are complementary to tryptic peptides [ 82 , 83 , 84 , 85 ].…”

Section: Mass Spectrometry-based Proteomics: Top-down Versus Middle-u...mentioning

confidence: 99%

How Can Proteomics Help to Elucidate the Pathophysiological Crosstalk in Muscular Dystrophy and Associated Multi-System Dysfunction?

Dowling,

Trollet,

Negroni

et al. 2024

Proteomes

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This perspective article is concerned with the question of how proteomics, which is a core technique of systems biology that is deeply embedded in the multi-omics field of modern bioresearch, can help us better understand the molecular pathogenesis of complex diseases. As an illustrative example of a monogenetic disorder that primarily affects the neuromuscular system but is characterized by a plethora of multi-system pathophysiological alterations, the muscle-wasting disease Duchenne muscular dystrophy was examined. Recent achievements in the field of dystrophinopathy research are described with special reference to the proteome-wide complexity of neuromuscular changes and body-wide alterations/adaptations. Based on a description of the current applications of top-down versus bottom-up proteomic approaches and their technical challenges, future systems biological approaches are outlined. The envisaged holistic and integromic bioanalysis would encompass the integration of diverse omics-type studies including inter- and intra-proteomics as the core disciplines for systematic protein evaluations, with sophisticated biomolecular analyses, including physiology, molecular biology, biochemistry and histochemistry. Integrated proteomic findings promise to be instrumental in improving our detailed knowledge of pathogenic mechanisms and multi-system dysfunction, widening the available biomarker signature of dystrophinopathy for improved diagnostic/prognostic procedures, and advancing the identification of novel therapeutic targets to treat Duchenne muscular dystrophy.

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“…One can therefore differentiate between in-solution [185,186], in-gel [187,188] and on-membrane [189][190][191] digestion protocols. The most frequently used protease in MS-based proteomics is trypsin [192], but alternative proteases can be used alone or in combination for protein digestion [193][194][195]. A rapid in-gel digestion protocol was recently designed for GeLC-MS/MS applications [196], which suits the systematic proteomic detection of very large proteins that do not properly move into the second dimension during 2D-GE [110].…”

Section: Protein Digestion For Peptide Mass Spectrometrymentioning

confidence: 99%

Fiber-Type Shifting in Sarcopenia of Old Age: Proteomic Profiling of the Contractile Apparatus of Skeletal Muscles

Dowling

Gargan

Swandulla

et al. 2023

IJMS

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The progressive loss of skeletal muscle mass and concomitant reduction in contractile strength plays a central role in frailty syndrome. Age-related neuronal impairments are closely associated with sarcopenia in the elderly, which is characterized by severe muscular atrophy that can considerably lessen the overall quality of life at old age. Mass-spectrometry-based proteomic surveys of senescent human skeletal muscles, as well as animal models of sarcopenia, have decisively improved our understanding of the molecular and cellular consequences of muscular atrophy and associated fiber-type shifting during aging. This review outlines the mass spectrometric identification of proteome-wide changes in atrophying skeletal muscles, with a focus on contractile proteins as potential markers of changes in fiber-type distribution patterns. The observed trend of fast-to-slow transitions in individual human skeletal muscles during the aging process is most likely linked to a preferential susceptibility of fast-twitching muscle fibers to muscular atrophy. Studies with senescent animal models, including mostly aged rodent skeletal muscles, have confirmed fiber-type shifting. The proteomic analysis of fast versus slow isoforms of key contractile proteins, such as myosin heavy chains, myosin light chains, actins, troponins and tropomyosins, suggests them as suitable bioanalytical tools of fiber-type transitions during aging.

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Protein Digestion for 2D-DIGE Analysis

Cited by 6 publications

References 50 publications

Proteomic reference map for sarcopenia research: mass spectrometric identification of key muscle proteins located in the sarcomere, cytoskeleton and the extracellular matrix

Proteomic reference map for sarcopenia research: mass spectrometric identification of key muscle proteins located in the sarcomere, cytoskeleton and the extracellular matrix

How Can Proteomics Help to Elucidate the Pathophysiological Crosstalk in Muscular Dystrophy and Associated Multi-System Dysfunction?

Fiber-Type Shifting in Sarcopenia of Old Age: Proteomic Profiling of the Contractile Apparatus of Skeletal Muscles

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