Microwave‐enhanced enzyme reaction for protein mapping by mass spectrometry: A new approach to protein digestion in minutes

Pramanik, Birendra N.; Mirza, Urooj A.; Ing, Yao Hain; Liu, Yan‐Hui; Bartner, Peter L.; Weber, Patricia C; Bose, Ajay K.

doi:10.1110/ps.0213702

Cited by 217 publications

(149 citation statements)

References 41 publications

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“…The mass difference between the measured molecular weights of the two isoforms is 1380 Da, which is close to the 1413 Da proposed by the translated protein sequences based on the two cDNAs. Previous characterization of Ara h 2 provided the identification of an N-terminal peptide [2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18] and two tryptic peptides [82][83][84][85][86][87][88][89][90][91][92][93][94] and [95][96][97][98][99][100][101][102][103][104][105][106][107][108][109][110], which were proposed as potential biomarkers for regulatory monitoring. 6 A recent study of 2-D gel electrophoretic patterns of proteins from the Virginia type peanut identified fourteen spots that were linked to Ara h 2 by partial peptide mass fingerprinting.…”

Section: Introductionmentioning

confidence: 99%

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Primary sequence and site‐selective hydroxylation of prolines in isoforms of a major peanut allergen protein Ara h 2

Shefcheck

Callahan

et al. 2010

Protein Science

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The Ara h 2 proteins are major determinants of peanut allergens. These proteins have not been fully studied at the molecular level. It has been previously proposed that there are two isoforms of Ara h 2, based on primary structures that were deduced from two reported cDNA sequences. In this report, four isoforms have been purified and characterized individually. Mass spectrometric methods have been used to determine the protein sequences and to define posttranslational modifications for all four isoforms. Two pairs of isoforms have been identified, corresponding to a long-chain form and a form that is shorter by 12 amino acids. Each pair is further differentiated by the presence or absence of a two amino acid sequence at the carboxyl terminus of the protein. Modifications that were characterized include site-specific hydroxylation of proline residues, but no glycosylation was found, in contrast to previous reports.

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Section: Introductionmentioning

confidence: 99%

“…Microwave acceleration was used to enhance the enzymatic (trypsin) proteolysis of the reduced isoforms. 9 Then the peptide products were analyzed using LC-MS/MS. Manual interpretation of the MS/MS spectra allowed de novo sequencing of the primary structures of all the isoforms of the Ara h 2 and identification and location of the PTM.…”

Section: Introductionmentioning

confidence: 99%

Primary sequence and site‐selective hydroxylation of prolines in isoforms of a major peanut allergen protein Ara h 2

Shefcheck

Callahan

et al. 2010

Protein Science

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“…This approach exploits unique microwave properties to accelerate the enzymatic digestion. In recent years, microwaveassisted proteolytic digestion procedures, including tryptic [6][7][8] and acid-mediated proteolysis [9][10][11][12], have enabled samples to be prepared more rapidly for bottom-up analysis.…”

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confidence: 99%

Digestion completeness of microwave-assisted and conventional trypsin-catalyzed reactions

Reddy

Hsu

et al. 2010

J. Am. Soc. Mass Spectrom.

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Microwave-assisted proteolytic digestion often yields misscleaved peptides, attributed to incomplete hydrolysis reactions between enzymes and substrates. The number of missed cleavages is an important parameter in proteome database searching. This study investigates how various factors affect digestion processes. Optimum conditions for microwave-assisted digestion (50 mM Tris buffer, 30 min at 60°C, and enzyme to protein molar ratio of 1:5) were determined. The digestion products obtained from eight standard proteins were characterized based on matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Experimental results indicate that the digestion temperature, reaction time, enzyme to substrate ratio, and digestion buffer affect the number of misscleaved peptides and incomplete digestion percentages. Although all protein molecules in a sample could be digested into peptides within a few minutes under microwave irradiation, longer reaction times or methods to maximize the enzyme activity should be considered if digestion completeness is a major concern. (J Am Soc Mass Spectrom 2010, 21, 421-424)

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“…For example, microwave-assisted [6] digestion, proposed by Pramanik et al in 2002 [7], successfully reduced the digestion time from hours to 10 min. Turapov et al reported a fast digestion method using a PCR-type thermocycler [8], with the caveat that many proteases cannot withstand the elevated temperatures.…”

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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Microwave‐enhanced enzyme reaction for protein mapping by mass spectrometry: A new approach to protein digestion in minutes

Cited by 217 publications

References 41 publications

Primary sequence and site‐selective hydroxylation of prolines in isoforms of a major peanut allergen protein Ara h 2

Primary sequence and site‐selective hydroxylation of prolines in isoforms of a major peanut allergen protein Ara h 2

Digestion completeness of microwave-assisted and conventional trypsin-catalyzed reactions

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

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Microwave‐enhanced enzyme reaction for protein mapping by mass spectrometry: A new approach to protein digestion in minutes

Cited by 217 publications

References 41 publications

Primary sequence and site‐selective hydroxylation of prolines in isoforms of a major peanut allergen protein Ara h 2

Primary sequence and site‐selective hydroxylation of prolines in isoforms of a major peanut allergen protein Ara h 2

Digestion completeness of microwave-assisted and conventional trypsin-catalyzed reactions

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