Protocol

Clinical Proteomics

Volume 428 of the series Methods in Molecular Biology™ pp 209-230

Label-Free LC-MS Method for the Identification of Biomarkers

  • Richard E. HiggsAffiliated withSchool of Life Sciences, University of Hertfordshire College LaneHatfield
  • , Michael D. KniermanAffiliated withSchool of Life Sciences, University of Hertfordshire College LaneHatfield
  • , Valentina GelfanovaAffiliated withSchool of Life Sciences, University of Hertfordshire College LaneHatfield
  • , Jon P. ButlerAffiliated withSchool of Life Sciences, University of Hertfordshire College LaneHatfield
  • , John E. HaleAffiliated withSchool of Life Sciences, University of Hertfordshire College LaneHatfield

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Summary

Pharmaceutical companies and regulatory agencies are pursuing biomarkers as a means to increase the productivity of drug development. Quantifying differential levels of proteins from complex biological samples like plasma or cerebrospinal fluid is one specific approach being used to identify markers of drug action, efficacy, toxicity, etc. Academic investigators are also interested in markers that are diagnostic or prognostic of disease states. We report a comprehensive, fully automated, and label-free approach to relative protein quantification including: sample preparation, proteolytic protein digestion, LC-MS/MS data acquisition, de-noising, mass and charge state estimation, chromatographic alignment, and peptide quantification via integration of extracted ion chromatograms. Additionally, we describe methods for transformation and normalization of the quantitative peptide levels in multiplexed measurements to improve precision for statistical analysis. Lastly, we outline how the described methods can be used to design and power biomarker discovery studies.

Key Words

relative quantification label-free quantification biomarkers proteomics LC-MS/MS