A multiple reaction monitoring method for absolute quantification of the human liver alcohol dehydrogenase ADH1C1 isoenzyme

• Published in: Analytical biochemistry Vol. 369; no. 1; pp. 18 - 26
• Main Authors: Janecki, Dariusz J., Bemis, Kerry G., Tegeler, Tony J., Sanghani, Paresh C., Zhai, Lanmin, Hurley, Thomas D., Bosron, William F., Wang, Mu
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01-10-2007
• Subjects: Absolute quantification; Alcohol dehydrogenase; Alcohol Dehydrogenase - analysis; Alcohol Dehydrogenase - metabolism; Amino Acid Sequence; Human liver; Humans; Isoenzymes - analysis; Isoenzymes - metabolism; Isotope Labeling - methods; LC–MS/MS; Liver - enzymology; Mass Spectrometry; Molecular Sequence Data; Multiple reaction monitoring; Peptides - chemistry; Peptides - metabolism; Proteomics - methods; Alcohol dehydrogenase; Multiple reaction monitoring; LC–MS/MS; Absolute quantification; Human liver
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1016/j.ab.2007.06.043

Although significant progress has been made in protein quantification using mass spectrometry during recent years, absolute protein quantification in complex biological systems remains a challenging task in proteomics. The use of stable isotope-labeled standard peptide is the most commonly used strategy for absolute quantification, but it might not be suitable in all instances. Here we report an alternative strategy that employs a stable isotope-labeled intact protein as an internal standard to absolutely quantify the alcohol dehydrogenase (ADH) expression level in a human liver sample. In combination with a new targeted proteomics approach employing the method of multiple reaction monitoring (MRM), we precisely and quantitatively measured the absolute protein expression level of an ADH isoenzyme, ADH1C1, in human liver. Isotope-labeled protein standards are predicted to be particularly useful for measurement of highly homologous isoenzymes such as ADHs where multiple signature peptides can be examined by MRM in a single experiment.