Overview on modern approaches to speed up protein identification workflows relying on enzymatic cleavage and mass spectrometry-based techniques

Capelo, J.L.; Carreira, Ricardo J.; Diniz, Mário; Fernandes, Luz; Galesio, M.; Lodeiro, Carlos; Santos, Hugo M.; Vale, Gonçalo

doi:10.1016/j.aca.2009.07.034

Cited by 89 publications

(69 citation statements)

References 41 publications

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“…The accelerated rate of digestion as well as a greater sequence coverage have been demonstrated in mass spectrometry analyses of single proteins or biological samples (1,8,20). High sequence coverages of standard proteins, i.e.…”

Section: Sequence Coverage and Detection Of Peptide Markersmentioning

confidence: 99%

“…The complexity of food matrices, processes of protein denaturation and aggregation as well as long duration of protein digestion are the main hurdles in the identifi cation of processed proteins and peptides in any traditional workfl ow. Recently, a number of new tools have been introduced to speed up protein identifi cation (1). The application of microspin columns, ultrasonic, infrared, microwave energy, high pressure or microreactors substantially reduced the sample preparation time, involving the steps of protein solubilization, reduction, alkylation, and digestion.…”

Section: Introductionmentioning

confidence: 99%

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Processed Meat Protein and Heat-Stable Peptide Marker Identification Workflow Using Microwave-Assisted Tryptic Digestion

Montowska

Pośpiech

2016

Food Technol. Biotechnol.

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SummaryNew approaches to rapid examination of proteins and peptides in complex food matrices are of great interest to the community of food scientists. The aim of the study is to examine the infl uence of microwave irradiation on the acceleration of enzymatic cleavage and enzymatic digestion of denatured proteins in cooked meat of fi ve species (catt le, horse, pig, chicken and turkey) and processed meat products (coarsely minced, smoked, cooked and semi-dried sausages). Severe protein aggregation occurred not only in heated meat under harsh treatment at 190 °C but also in processed meat products. All the protein aggregates were thoroughly hydrolyzed aft er 1 h of trypsin treatment with short exposure times of 40 and 20 s to microwave irradiation at 138 and 303 W. There were much more missed cleavage sites observed in all microwave-assisted digestions. Despite the incompleteness of microwave-assisted digestion, six unique peptide markers were detected, which allowed unambiguous identifi cation of processed meat derived from the examined species. Although the microwave-assisted tryptic digestion can serve as a tool for rapid and high-throughput protein identifi cation, great caution and pre-evaluation of individual samples is recommended in protein quantitation.

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Section: Sequence Coverage and Detection Of Peptide Markersmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Processed Meat Protein and Heat-Stable Peptide Marker Identification Workflow Using Microwave-Assisted Tryptic Digestion

Montowska

Pośpiech

2016

Food Technol. Biotechnol.

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“…In addition, procedures to enhance the protease activity, such as the application of microwaves [32], high pressure [33], or the energy produced by ultrasound [34], can accelerate the time consuming trypsin digestion. The application of only 1-2 minutes of High Intensity Focused Ultrasound (HIFU) to in-solution tryptic digestions has been reported to achieve an efficiency and reproducibility similar to that obtained by traditional overnight protocols [34][35][36].…”

Section: Accepted M Manuscriptmentioning

confidence: 99%

Rapid direct detection of the major fish allergen, parvalbumin, by selected MS/MS ion monitoring mass spectrometry

Carrera

Cañas

Gallardo³

2012

Journal of Proteomics

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“…The other approach utilizes heating the sample at 95 o C for 5 min, which can be reversible if the sample is not processed immediately. 9,11 Organic solvents as denaturing agents have also been used, but they have been shown to precipitate protein; hence, the concentration has to be carefully examined. Protein denaturation to unfold secondary and tertiary structures and to ensure the complete tryptic digestion is one of the rate limiting steps in sample preparation.…”

Section: -9mentioning

confidence: 99%

Effects of Heat Shock Treatment on Enzymatic Proteolysis for LC-MS/MS Quantitative Proteome Analysis

Arul¹,

Han²,

Jang³

et al. 2016

Mass Spectrometry Letters

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: Various efforts have been developed to improve sample preparation steps, which strongly depend on hands-on processes for accurate and sensitive quantitative proteome analysis. In this study, we carried out heating the sample prior to trypsin digestion using an instrument to improve the tryptic digestion process. The heat shock generated by the system efficiently denatured proteins in the sample and increased the reproducibility in quantitative proteomics based on peptide abundance measurements. To demonstrate the effectiveness of the protocol, three cell lines (A human lung cancer cell line (A549), a human embryonic kidney cell line (HEK293T), and a human colorectal cancer cell line (HCT-116)) were selected and the effect of heat shock was compared to that of normal tryptic digestion processes. The tryptic digests were desalted and analysed by LC-MS/MS, the results showed 57 and 36% increase in the number of identified unique peptides and proteins, respectively, than conventional digestion. Heat shock treated samples showed higher numbers of shorter peptides and peptides with low inter-sample variation among triplicate runs. Quantitative LC-MS/MS analysis of heat shock treated sample yielded peptides with smaller relative error percentage for the triplicate run when the peak areas were compared. Exposure of heat-shock to proteomic samples prior to proteolysis in conventional digestion process can increase the digestion efficiency of trypsin resulting in production of increased number of peptides eventually leading to higher proteome coverage.

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Overview on modern approaches to speed up protein identification workflows relying on enzymatic cleavage and mass spectrometry-based techniques

Cited by 89 publications

References 41 publications

Processed Meat Protein and Heat-Stable Peptide Marker Identification Workflow Using Microwave-Assisted Tryptic Digestion

Processed Meat Protein and Heat-Stable Peptide Marker Identification Workflow Using Microwave-Assisted Tryptic Digestion

Rapid direct detection of the major fish allergen, parvalbumin, by selected MS/MS ion monitoring mass spectrometry

Effects of Heat Shock Treatment on Enzymatic Proteolysis for LC-MS/MS Quantitative Proteome Analysis

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