Isotopic N,N-Dimethyl Leucine (iDiLeu) for Absolute Quantification of Peptides Using a Standard Curve Approach
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Quantitative proteomics studies require an absolute quantification step to accurately measure changes in protein concentration. Absolute quantification using liquid chromatography–mass spectrometry (LC–MS) traditionally combines triple quadrupole instrumentation with stable isotope-labeled standards to measure protein concentrations via their enzymatically produced peptides. Chemical modification of peptides using labels like mass differential tags for relative and absolute quantification (mTRAQ) provides another route to determine protein quantities. This chapter describes a cost-effective and high-throughput chemical labeling method that utilizes five amine-reactive, isotopic N,N-dimethyl leucine (iDiLeu) reagents. These tags enable generation of four-point calibration curves in one LC–MS run to determine protein concentrations from labeled peptides. In particular, we provide a detailed workflow for protein quantification using the iDiLeu reagent that includes important considerations like labeling conditions and isotopic interference correction.
Key wordsQuantification iDiLeu DiLeu Mass difference labeling Proteomics Calibration curve
The authors acknowledge support for this work by the National Institutes of Health grant (1R01DK071801). The Q-Exactive Orbitrap was purchased through the support of an NIH shared instrument grant (NIH-NCRR S10RR029531). L.L acknowledges an H.I. Romnes Faculty Research Fellowship.