Relative Quantification of Proteins in Human Cerebrospinal Fluids by MS/MS Using 6-Plex Isobaric Tags

Abstract

A new 6-plex isobaric mass tagging technology is presented, and proof of principle studies are carried out using standard protein mixtures and human cerebrospinal fluid (CSF) samples. The Tandem Mass Tags (TMT) comprise a set of structurally identical tags which label peptides on free amino-terminus and epsilon-amino functions of lysine residues. During MS/MS fragmentation, quantification information is obtained through the losses of the reporter ions. After evaluation of the relative quantification with the 6-plex version of the TMT on a model protein mixture at various concentrations, the quantification of proteins in CSF samples was performed using shotgun methods. Human postmortem (PM) CSF was taken as a model of massive brain injury and comparison was carried out with antemortem (AM) CSF. After immunoaffinity depletion, triplicates of AM and PM CSF pooled samples were reduced, alkylated, digested by trypsin, and labeled, respectively, with the six isobaric variants of the TMT (with reporter ions from m/z = 126.1 to 131.1 Th). The samples were pooled and fractionated by SCX chromatography. After RP-LC separation, peptides were identified and quantified by MS/MS analysis with MALDI TOF/TOF and ESI-Q-TOF. The concentration of 78 identified proteins was shown to be clearly increased in PM CSF samples compared to AM. Some of these proteins, like GFAP, protein S100B, and PARK7, have been previously described as brain damage biomarkers, supporting the PM CSF as a valid model of brain insult. ELISA for these proteins confirmed their elevated concentration in PM CSF. This work demonstrates the validity and robustness of the tandem mass tag (TMT) approach for quantitative MS-based proteomics.

Addressing Accuracy and Precision Issues in iTRAQ Quantitation

“…There is significant interest in stable isotope labeling strategies of proteins or peptides as with every measurement there is the potential to use an internal reference allowing relative quantitation comparison, which significantly increases sensitivity of detection of change in abundance. Isobaric labeling techniques such as tandem mass tags (11,12) or isobaric tags for relative or absolute quantitation (iTRAQ) 1 (13,14) allow multiplexing of four, six and eight separately labeled samples within one experiment. In contrast to most other quantitative proteomics methods where precursor ion intensities are measured, here the measurement and ensuing quantitation of iTRAQ reporter ions occurs after fragmentation of the precursor ion.…”

Proteomic techniques to probe the ubiquitin landscape

“…Both SILAC and dimethylation increase sample complexity and compare analyte intensities at the MS1 level. A more sophisticated chemical labeling approach uses reagents such as iTRAQ [46] and TMT [47], which permit a greater number of channels to be monitored simultaneously.…”

Proteomic Approaches and Identification of Novel Therapeutic Targets for Alcoholism

“…Labeling techniques use isotopic labels as a reference for relative or absolute quantitation. These labels can be introduced into samples in vivo by metabolic labeling (Wu et al, 2004) or SILAC (Ong et al, 2002), and in vitro by iTRAQ (Ross et al, 2004) or TMT tags (Dayon et al, 2008). In particular, iTRAQ is a comprehensive and efficient method for proteomic quantification that provides multiplex capability and can identify lowexpressed proteins with high confidence in complex samples.…”