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Peptide quantification assay

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The Peptide Quantification Assay is a laboratory equipment used to quantify the concentration of peptides in a sample. It provides accurate measurement of peptide levels, enabling researchers to analyze and characterize these important biomolecules.

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Lab products found in correlation

Qubit protein quantification assay, by Thermo Fisher Scientific (3 mentions) 11 plex tandem mass tag reagents, by Thermo Fisher Scientific (2 mentions) High ph reverse phase peptide fractionation, by Thermo Fisher Scientific (2 mentions) S trap micro column, by Protifi (2 mentions) Rapigest, by Waters Corporation (1 mentions)

3 protocols using peptide quantification assay

1

Subcellular Protein Fractionation and TMT Labeling

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5x106 frozen cell pellets from each condition (n=5) were used to perform subcellular protein fractionation. Minute Cytoplasmic and Nuclear Extraction kit (Invent Biotech, MN) was used to prepare nuclear and cytoplasmic fractions according to the manufacturer’s instruction. Protein concentration was measured by Qubit Protein Quantification Assay (Thermo Fisher, Rockford, IL). 35μg of protein lysate (nuclear or cytoplasmic) from each sample were trapped and digested by trypsin on a micro S-trap column (PROTIFI, NY) according to the manufacturer’s instruction. 0.6μg of the peptide mixture was used for peptide quantification assay (Thermo Fisher, Rockford, IL).
Isobaric labeling of peptides was performed using the 11-plex tandem mass tag (TMT) reagents (Thermo Fisher Scientific, Rockford, IL). TMT reagents (0.8mg) were dissolved in 41μl of dry acetonitrile (ACN), and 20.5μl was added to 25μg of digested peptide in 100mM TEAB. After 1 hour incubation at room temperature, the reaction was quenched by adding 4.2μl of 5% hydroxylamine. Labeled peptides were combined and dried for the subsequent high pH reverse phase peptide fractionation (Thermo Fisher, Rockford, IL). 8 fractions were generated for each combined set. Each TMT labeled fraction was reconstituted in a solution of 2 % acetonitrile (ACN), 2 % formic acid (FA) for MS analysis.
Yan H., Yu C.C., Fine S.A., Youssof A.L., Yang Y.R., Yan J., Karg D.C., Cheung E.C., Friedman R.A., Ying H., Chen E.I., Luo J., Miao Y., Qiu W, & Su G.H. (2021). Loss of the Wild-type KRAS Allele Promotes Pancreatic Cancer Progression through Functional Activation of YAP1. Oncogene, 40(50), 6759-6771.
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2

Quantitative Proteomics by TMT10-plex Labeling

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Each cell pellet was homogenized in lysis buffer (4 M Urea in 50 mM Ammonium Bicarbonate, 0.1% RapiGestTM; Waters, MA) and Roche protease inhibitor cocktail) and protein concentration was measured by the Qubit Protein Quantification Assay (Thermo Fisher Scientific, Rockford, IL). An equal amount of protein lysate from each sample was pooled as a common reference to generate ratios in the TMT10plex labeling experiment. 15 μg of protein lysates were reduced with 10 mM TCEP and alkylated with iodoacetamide (18.75 mM) in the dark for 30 minute. Protein was precipitated by the methanol-chloroform protocol and re-suspended in 100 mM TEAB. 375 ng of trypsin was added to the protein extract in a 1:40 enzyme to protein ratio and incubated for 16 hours at 37 °C. Next day, samples were centrifuged for 30 min at 14,000 rpm to remove insoluble materials and 0.6 μg of the peptide mixture was used for peptide quantification assay (Thermo Fisher Scientific).
Lee Y., Pujol Canadell M., Shuryak I., Perrier J.R., Taveras M., Patel P., Koller A., Smilenov L.B., Brenner D.J., Chen E.I, & Turner H.C. (2018). Candidate protein markers for radiation biodosimetry in the hematopoietically humanized mouse model. Scientific Reports, 8, 13557.
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3

Subcellular Protein Fractionation and TMT Labeling

Check if the same lab product or an alternative is used in the 5 most similar protocols
5x106 frozen cell pellets from each condition (n=5) were used to perform subcellular protein fractionation. Minute Cytoplasmic and Nuclear Extraction kit (Invent Biotech, MN) was used to prepare nuclear and cytoplasmic fractions according to the manufacturer’s instruction. Protein concentration was measured by Qubit Protein Quantification Assay (Thermo Fisher, Rockford, IL). 35μg of protein lysate (nuclear or cytoplasmic) from each sample were trapped and digested by trypsin on a micro S-trap column (PROTIFI, NY) according to the manufacturer’s instruction. 0.6μg of the peptide mixture was used for peptide quantification assay (Thermo Fisher, Rockford, IL).
Isobaric labeling of peptides was performed using the 11-plex tandem mass tag (TMT) reagents (Thermo Fisher Scientific, Rockford, IL). TMT reagents (0.8mg) were dissolved in 41μl of dry acetonitrile (ACN), and 20.5μl was added to 25μg of digested peptide in 100mM TEAB. After 1 hour incubation at room temperature, the reaction was quenched by adding 4.2μl of 5% hydroxylamine. Labeled peptides were combined and dried for the subsequent high pH reverse phase peptide fractionation (Thermo Fisher, Rockford, IL). 8 fractions were generated for each combined set. Each TMT labeled fraction was reconstituted in a solution of 2 % acetonitrile (ACN), 2 % formic acid (FA) for MS analysis.
Yan H., Yu C.C., Fine S.A., Youssof A.L., Yang Y.R., Yan J., Karg D.C., Cheung E.C., Friedman R.A., Ying H., Chen E.I., Luo J., Miao Y., Qiu W, & Su G.H. (2021). Loss of the Wild-type KRAS Allele Promotes Pancreatic Cancer Progression through Functional Activation of YAP1. Oncogene, 40(50), 6759-6771.
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