Addressing the Protease Bias in Quantitative Proteomics

• Protein quantification strategies using multiple proteases have been shown to deliver poor interprotease accuracy in label-free mass spectrometry experiments. By utilizing six different proteases with different cleavage sites, this study explores the protease bias and its effect on accuracy and precision by using recombinant protein standards. We established 557 SRM assays, using a recombinant protein standard resource, toward 10 proteins in human plasma and determined their concentration with multiple proteases. The quantified peptides of these plasma proteins spanned 3 orders of magnitude (0.02-70 μM). In total, 60 peptides were used for absolute quantification and the majority of the peptides showed high robustness. The retained reproducibility was achieved by quantifying plasma proteins using spiked stable isotope standard recombinant proteins in a targeted proteomics workflow. 

Ämnesord

MEDICIN OCH HÄLSOVETENSKAP  -- Medicinska och farmaceutiska grundvetenskaper -- Cell- och molekylärbiologi (hsv//swe)

MEDICAL AND HEALTH SCIENCES  -- Basic Medicine -- Cell and Molecular Biology (hsv//eng)

Nyckelord

absolute quantification

multiple proteases

plasma proteomics

SRM

targeted proteomics

Blood Proteins

Endopeptidases

Humans

Isotope Labeling

Isotopes

Peptide Hydrolases

Peptides

Proteomics

Recombinant Proteins

Reproducibility of Results

peptide

plasma protein

proteinase

recombinant protein

stable isotope

isotope

peptide hydrolase

Article

controlled study

female

human

kernel method

male

mass spectrometry

plasma

quantitative analysis

reproducibility

procedures

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