Comprehensive label-free method for the relative quantification of proteins from biological samples.
Pharmaceutical companies and regulatory agencies are broadly pursuing biomarkers as a means to increase the productivity of drug development. Quantifying differential levels of proteins from complex biological samples such as plasma or cerebrospinal fluid is one specific approach being used to identify markers of drug action, efficacy, toxicity, etc. We have developed a comprehensive, fully automated, and label-free approach to relative protein quantification from LC-MS/MS experiments of proteolytic protein digests including: de-noising, mass and charge state estimation, chromatographic alignment, and peptide quantification via integration of extracted ion chromatograms. Results from a variance components study of the entire method indicate that most of the variability is attributable to the LC-MS injection, with a median peptide LC-MS injection coefficient of variation of 8% on a ThermoFinnigan LTQ mass spectrometer. Spiked recovery results suggest a quantifiable range of approximately 32-fold for a sample protein.
Higgs
Richard E
RE
Lilly Research Laboratories, MS 1533, Lilly Corporate Center, Indianapolis, IN 46285, USA. higgs@lilly.com
Knierman
Michael D
MD
Gelfanova
Valentina
V
Butler
Jon P
JP
Hale
John E
JE
D000595:Amino Acid Sequence; D000818:Animals; D015415:Biomarkers; D002853:Chromatography, Liquid / Q000379:methods*; D008297:Male; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D011506:Proteins / Q000032:analysis*; D051381:Rats; D015203:Reproducibility of Results; D044967:Serum / Q000737:chemistry; D014360:Trypsin Inhibitor, Kunitz Soybean / Q000032:analysis
D015415:Biomarkers; D011506:Proteins; D014360:Trypsin Inhibitor, Kunitz Soybean