A method to evaluate tryptic digestion efficiency for high‐throughput proteome analyses

Wierenga, Susan K.; Zocher, Mark J.; Mirus, Matthew M.; Conrads, Thomas P.; Goshe, Michael B.; Veenstra, Timothy D.

doi:10.1002/rcm.729

Cited by 23 publications

(14 citation statements)

References 10 publications

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“…The decision to optimize these factors is determined by whether complete digestion is required for the study. In traditional proteomics, protein identification benefits greatly from ensuring complete proteolytic digestion across the proteome [18]; however, the targeted proteomics approach may only require complete cleavage of the surrogate peptides. Therefore, these factors were not evaluated in this study.…”

Section: Digestion Efficiencymentioning

confidence: 99%

Quantification of human serum transferrin using liquid chromatography–tandem mass spectrometry based targeted proteomics

Liu

et al. 2012

Journal of Chromatography B

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Section: Digestion Efficiencymentioning

confidence: 99%

Quantification of human serum transferrin using liquid chromatography–tandem mass spectrometry based targeted proteomics

Liu

et al. 2012

Journal of Chromatography B

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“…Synthetic unlabeled and 13 C-, 15 N-labeled proteotypic peptides (Table 1) were ordered from Thermo Scientific (Ulm, Germany). Peptide purity (.94%) was provided by the manufacturer, using RP-HPLC UV (with a detection wavelength at 215 nm) analysis and MALDI-TOF MS analysis.…”

Section: Synthetic Proteotypic Peptidesmentioning

confidence: 99%

“…In-solution digestion of recombinant P450s and HLM was performed using a protocol modified from the "denatured and reduced" method [15] and Pierce Biotechnology (product #89895; Rockford, IL). The final protocol were optimized with respect to the length of protein denaturation by heat and tryptic digestion, the requirement of IAA alkylation, the amount of trypsin used, and the effect of detergent Brij 35.…”

Section: In-solution Digestion and Quantification Via Lc-ms/ms Analysismentioning

confidence: 99%

“…Upon alkylation, 1 mg of trypsin was spiked into the reduced and alkylated protein sample (100 mL), and digestion was carried out at 377C for 4 h with shaking midway during incubation. Digestion was quenched with a half-volume of ice-cold ACN spiked with 13 C-, 15 N-labeled proteotypic peptides as IS. After mixing, the sample was centrifuged at ,14006g for 10 min, and 8 mL of supernatant was injected for LC-MS/MS analysis.…”

Section: In-solution Digestion and Quantification Via Lc-ms/ms Analysismentioning

confidence: 99%

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A gel‐free MS‐based quantitative proteomic approach accurately measures cytochrome P450 protein concentrations in human liver microsomes

Wang

Dennison

et al. 2008

Proteomics

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The human cytochrome P450 (P450) superfamily consists of membrane-bound proteins that metabolize a myriad of xenobiotics and endogenous compounds. Quantification of P450 expression in various tissues under normal and induced conditions has an important role in drug safety and efficacy. Conventional immunoquantification methods have poor dynamic range, low throughput, and a limited number of specific antibodies. Recent advances in MS-based quantitative proteomics enable absolute protein quantification in a complex biological mixture. We have developed a gel-free MS-based protein quantification strategy to quantify CYP3A enzymes in human liver microsomes (HLM). Recombinant protein-derived proteotypic peptides and synthetic stable isotope-labeled proteotypic peptides were used as calibration standards and internal standards, respectively. The lower limit of quantification was approximately 20 fmol P450. In two separate panels of HLM examined (n = 11 and n = 22), CYP3A, CYP3A4 and CYP3A5 concentrations were determined reproducibly (CV or=0.87) and marker activities (r(2)>or=0.88), including testosterone 6beta-hydroxylation (CYP3A), midazolam 1'-hydroxylation (CYP3A), itraconazole 6-hydroxylation (CYP3A4) and CYP3A5-mediated vincristine M1 formation (CYP3A5). Taken together, our MS-based method provides a specific, sensitive and reliable means of P450 protein quantification and should facilitate P450 characterization during drug development, especially when specific substrates and/or antibodies are unavailable.

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“…After cooling to room temperature, the sample was reduced using 5 mM dithiothreitol with heating at 56°C for 1 h followed by alkylation using 10 mM iodoacetamide at 25°C for 1 h. Trypsin was added to the reduced and alkylated LMW filtrate at a protein-to-enzyme ratio of 50:1, followed by incubation overnight at 37°C. While no chemical denaturant was added to the sample, the long incubation time should allow for a sufficiently high digestion efficiency that produces a complex mixture of peptides by which to characterize proteins within the LMW serum fraction (14). The digestion was stopped by acidifying with trifluoroacetic acid to a final concentration of 0.1% and desalted using a Bond Elut C-18 reversed-phase solid-phase extraction column as per the manufacturer's protocol.…”

mentioning

confidence: 99%

Characterization of the Low Molecular Weight Human Serum Proteome

Tirumalai

Chan

Prieto

et al. 2003

Molecular & Cellular Proteomics

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742

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Serum potentially carries an archive of important histological information whose determination could serve to improve early disease detection. The analysis of serum, however, is analytically challenging due to the high dynamic concentration range of constituent protein/peptide species, necessitating extensive fractionation prior to mass spectrometric analyses. The low molecular weight (LMW) serum proteome is that protein/peptide fraction from which high molecular weight proteins, such as albumin, immunoglobulins, transferrin, and lipoproteins, have been removed. This LMW fraction is made up of several classes of physiologically important proteins such as cytokines, chemokines, peptide hormones, as well as proteolytic fragments of larger proteins. Centrifugal ultrafiltration of serum was used to remove the large constituent proteins resulting in the enrichment of the LMW proteins/ peptides. Because albumin is known to bind and transport small molecules and peptides within the circulatory system, the centrifugal ultrafiltration was conducted under solvent conditions effecting the disruption of protein-protein interactions. The LMW serum proteome sample was digested with trypsin, fractionated by strong cation exchange chromatography, and analyzed by microcapillary reversed-phase liquid chromatography coupled on-line with electrospray ionization tandem mass spectrometry. Analysis of the tandem mass spectra resulted in the identification of over 340 human serum proteins; however, not a single peptide from serum albumin was observed. The large number of proteins identified demonstrates the efficacy of this method for the removal of large abundant proteins and the enrichment of the LMW serum proteome.

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A method to evaluate tryptic digestion efficiency for high‐throughput proteome analyses

Cited by 23 publications

References 10 publications

Quantification of human serum transferrin using liquid chromatography–tandem mass spectrometry based targeted proteomics

Quantification of human serum transferrin using liquid chromatography–tandem mass spectrometry based targeted proteomics

A gel‐free MS‐based quantitative proteomic approach accurately measures cytochrome P450 protein concentrations in human liver microsomes

Characterization of the Low Molecular Weight Human Serum Proteome

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