title: Hyperplex-MRM: a hybrid multiple reaction monitoring method using mTRAQ/iTRAQ labeling for multiplex absolute quantification of human colorectal cancer biomarker.
abstract: Novel biomarker verification assays are urgently required to improve the efficiency of biomarker development. Benefitting from lower development costs, multiple reaction monitoring (MRM) has been used for biomarker verification as an alternative to immunoassay. However, in general MRM analysis, only one sample can be quantified in a single experiment, which restricts its application. Here, a Hyperplex-MRM quantification approach, which combined mTRAQ for absolute quantification and iTRAQ for relative quantification, was developed to increase the throughput of biomarker verification. In this strategy, equal amounts of internal standard peptides were labeled with mTRAQ reagents Δ0 and Δ8, respectively, as double references, while 4-plex iTRAQ reagents were used to label four different samples as an alternative to mTRAQ Δ4. From the MRM trace and MS/MS spectrum, total amounts and relative ratios of target proteins/peptides of four samples could be acquired simultaneously. Accordingly, absolute amounts of target proteins/peptides in four different samples could be achieved in a single run. In addition, double references were used to increase the reliability of the quantification results. Using this approach, three biomarker candidates, ademosylhomocysteinase (AHCY), cathepsin D (CTSD), and lysozyme C (LYZ), were successfully quantified in colorectal cancer (CRC) tissue specimens of different stages with high accuracy, sensitivity, and reproducibility. To summarize, we demonstrated a promising quantification method for high-throughput verification of biomarker candidates.
authors.affiliation: Shanghai Cancer Center and Institutes of Biomedical Sciences, Fudan University, Shanghai 200032, PR China.
authors.forename: Hong-Rui
authors.initials: HR
authors.lastname: Yin
authors.forename: Lei
authors.initials: L
authors.lastname: Zhang
authors.forename: Li-Qi
authors.initials: LQ
authors.lastname: Xie
authors.forename: Li-Yong
authors.initials: LY
authors.lastname: Huang
authors.forename: Ye
authors.initials: Y
authors.lastname: Xu
authors.forename: San-Jun
authors.initials: SJ
authors.lastname: Cai
authors.forename: Peng-Yuan
authors.initials: PY
authors.lastname: Yang
authors.forename: Hao-Jie
authors.initials: HJ
authors.lastname: Lu
mesh_terms: D043383:Adenosylhomocysteinase / Q000737:chemistry / Q000378:metabolism; D000328:Adult; D000368:Aged; D000595:Amino Acid Sequence; D014408:Biomarkers, Tumor / Q000378:metabolism*; D002138:Calibration; D002402:Cathepsin D / Q000737:chemistry / Q000378:metabolism; D015179:Colorectal Neoplasms / Q000175:diagnosis / Q000378:metabolism*; D005260:Female; D006801:Humans; D008297:Male; D008875:Middle Aged; D009113:Muramidase / Q000737:chemistry / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry; D012015:Reference Standards; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013194:Staining and Labeling; D053719:Tandem Mass Spectrometry / Q000592:standards*
chemical_list: D014408:Biomarkers, Tumor; D010446:Peptide Fragments; D009113:Muramidase; C514728:lysozyme C, human; D043383:Adenosylhomocysteinase; C533922:CTSD protein, human; D002402:Cathepsin D