Protein Digestion: An Overview of the Available Techniques and Recent Developments

Switzar, Linda; Giera, Martin; Niessen, W.M.A.

doi:10.1021/pr301201x

Cited by 203 publications

(188 citation statements)

References 133 publications

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“…Therefore, the use of alternative enzymes or combined proteolytic approaches (Figs. 5A-5D) to generate smaller peptide fragments containing the various PTMs of interest should improve the characterization and quantification of C-terminal single and multiple PTMs within this region via mass spectrometry (76). For example, the overall sequence coverage achieved by Glu-C proteolysis enables the unambiguous identification of all pathologically relevant PTMs (namely, pY125 or pS129, nY133, and other nitrated C-terminal Tyr residues) typically found within the C-terminal stretch of ␣-syn.…”

mentioning

confidence: 99%

Alpha-synuclein Post-translational Modifications as Potential Biomarkers for Parkinson Disease and Other Synucleinopathies

Schmid

Fauvet

Moniatte

et al. 2013

Molecular & Cellular Proteomics

176

145

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The development of novel therapies against neurodegenerative disorders requires the ability to detect their early, presymptomatic manifestations in order to enable treatment before irreversible cellular damage occurs. Precocious signs indicative of neurodegeneration include characteristic changes in certain protein levels, which can be used as diagnostic biomarkers when they can be detected in fluids such as blood plasma or cerebrospinal fluid. In the case of synucleinopathies, cerebrospinal alpha-synuclein (␣-syn) has attracted great interest as a potential biomarker; however, there is ongoing debate regarding the association between cerebrospinal ␣-syn levels and neurodegeneration in Parkinson disease and synucleinopathies. Post-translational modifications (PTMs) have emerged as important determinants of ␣-syn's physiological and pathological functions. Several PTMs are enriched within Lewy bodies and exist at higher levels in ␣-synucleinopathy brains, suggesting that certain modified forms of ␣-syn might be more relevant biomarkers than the total ␣-syn levels. However, the quantification of PTMs in bodily fluids poses several challenges. This review describes the limitations of current immunoassay-based ␣-syn quantification methods and highlights how these limitations can be overcome using novel mass-spectrometrybased assays. In addition, we describe how advances in chemical synthesis, which have enabled the preparation of ␣-syn proteins that are site-specifically modified at single or multiple residues, can facilitate the development of more accurate assays for detecting and quantifying ␣-syn PTMs in health and disease. Molecular & Cellular Proteomics

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mentioning

confidence: 99%

Alpha-synuclein Post-translational Modifications as Potential Biomarkers for Parkinson Disease and Other Synucleinopathies

Schmid

Fauvet

Moniatte

et al. 2013

Molecular & Cellular Proteomics

176

145

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“…Missed cleavages can result inconsistent production of the signature peptide in turn impacting quantitation. Peptides containing ragged ends or dibasic ends next to each other (such as in Arg-Arg, Lys-Lys, or Arg-Lys) should be avoided as they are known to result in missed cleavages [8][9][10].…”

Section: Signature Peptide Selectionmentioning

confidence: 99%

“…Urea is the most commonly used for denaturation during protein quantification. Alternatively, denaturation has been achieved using other chaotropic agents such as guanidine HCl, surfactants such as sodium deoxycholate, organic solvents such as methanol and heat (95 C) [13,17,26,41,42]. RapiGest SF, an acidlabile surfactant, is a Waters proprietary product that has gained high popularity for protein bioanalysis due to its compatibility with mass spectrometric detectors.…”

Section: Enzymatic Proteolysismentioning

confidence: 99%

“…Eight internal standardization approaches were compared with respect to accuracy and precision in work flows with and without digestion. Both analogue IS standard proteins (eel and human calcitonin), SIL-salmon calcitonin, SIL-salmon calcitonin signature peptide [1][2][3][4][5][6][7][8][9][10][11], and the cleavable SIL-salmon calcitonin peptide [1][2][3][4][5][6][7][8][9][10][11] were commercially obtained. 18 O-labeled form of the signature peptide was synthesized in-house by isotope exchange with18 O-labeled water.…”

Section: Comparison Of Protein Sil-is Versus Peptides Ismentioning

confidence: 99%

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Internal Standards for Absolute Quantification of Large Molecules (Proteins) from Biological Matrices by LC-MS/MS

Faria¹,

Halquist²

2018

Calibration and Validation of Analytical Methods - A Sampling of Current Approaches

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Internal standardization plays a critical role in the performance of a bioanalytical method. There has been a tremendous increase in the popularity of using liquid chromatography tandem mass spectrometry (LC-MS/MS) methods for quantitative bioanalysis of protein molecules. Protein, being too large to be directly analyzed by LC-MS/MS, is proteolyzed and a characteristic peptide is used as a surrogate analyte for quantification. Internal standardization in small molecules' analysis is straightforward, i.e., either a stable labeled isotope (SIL) form of the analyte or a structural analogue is used. As protein quantification involves protein digestion to yield peptides, there are more options for internal standardization. Currently, internal standard selection is based on the availability of the internal standards and the sample preparation workflow. A SIL-form of the analyte protein is the ideal internal standard. However, its use is limited due to cost and commercial availability. Alternatively, a SIL form the surrogate peptide analyte or a cleavable SIL-peptide can be used as an IS. For preclinical bioanalysis of humanized IgG antibody-based drugs, a universal SIL analogue protein has been effectively used as an internal standard. This chapter focuses on internal standardization for the quantitative analysis of proteins, such as biotherapeutics and biomarkers, using LC-MS/MS.

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“…To accelerate the in-solution proteolytic digestion process, a number of approaches have been developed [4,5]. For example, microwave-assisted [6] digestion, proposed by Pramanik et al in 2002 [7], successfully reduced the digestion time from hours to 10 min.…”

Section: Introductionmentioning

confidence: 99%

Proteolytic Digestion and TiO₂Phosphopeptide Enrichment Microreactor for Fast MS Identification of Proteins

Deng

Lazar

2016

J. Am. Soc. Mass Spectrom.

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Abstract. The characterization of phosphorylation state(s) of a protein is best accomplished by using isolated or enriched phosphoprotein samples or their corresponding phosphopeptides. The process is typically time-consuming as, often, a combination of analytical approaches must be used. To facilitate throughput in the study of phosphoproteins, a microreactor that enables a novel strategy for performing fast proteolytic digestion and selective phosphopeptide enrichment was developed. The microreactor was fabricated using 100 μm i.d. fused-silica capillaries packed with 1-2 mm beds of C18 and/or TiO 2 particles. Proteolytic digestion-only, phosphopeptide enrichment-only, and sequential proteolytic digestion/phosphopeptide enrichment microreactors were developed and tested with standard protein mixtures. The protein samples were adsorbed on the C18 particles, quickly digested with a proteolytic enzyme infused over the adsorbed proteins, and further eluted onto the TiO 2 microreactor for enrichment in phosphopeptides. A number of parameters were optimized to speed up the digestion and enrichments processes, including microreactor dimensions, sample concentrations, digestion time, flow rates, buffer compositions, and pH. The effective time for the steps of proteolytic digestion and enrichment was less than 5 min. For simple samples, such as standard protein mixtures, this approach provided equivalent or better results than conventional bench-top methods, in terms of both enzymatic digestion and selectivity. Analysis times and reagent costs were reduced~10-to 15-fold. Preliminary analysis of cell extracts and recombinant proteins indicated the feasibility of integration of these microreactors in more advanced workflows amenable for handling real-world biological samples.

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Protein Digestion: An Overview of the Available Techniques and Recent Developments

Cited by 203 publications

References 133 publications

Alpha-synuclein Post-translational Modifications as Potential Biomarkers for Parkinson Disease and Other Synucleinopathies

Alpha-synuclein Post-translational Modifications as Potential Biomarkers for Parkinson Disease and Other Synucleinopathies

Internal Standards for Absolute Quantification of Large Molecules (Proteins) from Biological Matrices by LC-MS/MS

Proteolytic Digestion and TiO₂Phosphopeptide Enrichment Microreactor for Fast MS Identification of Proteins

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Protein Digestion: An Overview of the Available Techniques and Recent Developments

Cited by 203 publications

References 133 publications

Alpha-synuclein Post-translational Modifications as Potential Biomarkers for Parkinson Disease and Other Synucleinopathies

Alpha-synuclein Post-translational Modifications as Potential Biomarkers for Parkinson Disease and Other Synucleinopathies

Internal Standards for Absolute Quantification of Large Molecules (Proteins) from Biological Matrices by LC-MS/MS

Proteolytic Digestion and TiO2Phosphopeptide Enrichment Microreactor for Fast MS Identification of Proteins

Contact Info

Product

Resources

About

Proteolytic Digestion and TiO₂Phosphopeptide Enrichment Microreactor for Fast MS Identification of Proteins