Differential protein expression analysis using stable isotope labeling and PQD linear ion trap MS technology

Abstract

An isotope tags for relative and absolute quantitation (iTRAQ)-based reversed-phase liquid chromatography (RPLC)-tandem mass spectrometry (MS/MS) method was developed for differential protein expression profiling in complex cellular extracts. The estrogen positive MCF-7 cell line, cultured in the presence of 17␤-estradiol (E2) and tamoxifen (Tam), was used as a model system. MS analysis was performed with a linear trap quadrupole (LTQ) instrument operated by using pulsed Q dissociation (PQD) detection. Optimization experiments were conducted to maximize the iTRAQ labeling efficiency and the number of quantified proteins. MS data filtering criteria were chosen to result in a false positive identification rate of Ͻ4%. The reproducibility of protein identifications was ϳ60%-67% between duplicate, and ϳ50% among triplicate LC-MS/MS runs, respectively. The run-to-run reproducibility, in terms of relative standard deviations (RSD) of global mean iTRAQ ratios, was better than 10%. The quantitation accuracy improved with the number of peptides used for protein identification. From a total of 530 identified proteins (P Ͻ 0.001) in the E2/Tam treated MCF-7 cells, a list of 255 proteins (quantified by at least two peptides) was generated for differential expression analysis. A method was developed for the selection, normalization, and statistical evaluation of such datasets. An approximate ϳ2-fold change in protein expression levels was necessary for a protein to be selected as a biomarker candidate. According to this data processing strategy, ϳ16 proteins involved in biological processes such as apoptosis, RNA processing/metabolism, DNA replication/transcription/repair, cell proliferation and metastasis, were found to be up-or down-regulated. R ecently, two-dimensional liquid chromatography (2DLC) with tandem MS detection has emerged as an attractive technology for quantitative proteomic profiling of complex cellular extracts [1][2][3][4]. Stable isotope labeling and label-free quantitation strategies have been explored [4 -13]. Isotope labeling approaches rely on the covalent attachment of stable isotope tags to specific amino acid residues of proteins or peptides during metabolic, enzymatic, or chemical processes. Label-free quantitation methods rely on measuring peak areas, intensities, or spectral counts, and benefit from not having to chemically alter the sample. Throughput, however, is lower, and quantitation errors are higher, as the samples are processed independently. Among chemical labeling techniques, isotope tags for relative and absolute quantitation (iTRAQ) has received much attention [14 -30]. In this approach, peptides are labeled with isobaric tags at the N-terminus and the lysine side chains. MS/MS fragmentation produces signature ions that can be used to obtain quantitative information. Perhaps the most attractive advantage of this approach is that it can be used for the simultaneous quantification (relative or absolute) of up to four/eight different samples. Simplicity, of course, is an added benefit.A...

Quantitative Nuclear Proteomics Identifies mTOR Regulation of DNA Damage Response

“…We first described proof-of-principle studies demonstrating the potential for this method, which offers the powerful combination of multiplexed stable isotope labeling with a highly sensitive mass spectrometer (18,25). Given the relatively small number of studies to date using the ion trap instrumentation for iTRAQ studies (47,48), our results provide an important demonstration of the effectiveness of this method for large scale analysis within a complex sample, supporting its value as a general tool for quantitative proteomics studies.…”

Strategies and Challenges in Measuring Protein Abundance Using Stable Isotope Labeling and Tandem Mass Spectrometry

“…However, iTRAQ and TMT is limited to instruments that can provide good MS2 spectra in the 100-120 Da range, such as the QSTAR Quadrupole Time-of-Flight instrument (ABI). Pulsed Q dissociation (PQD) and higher energy C-trap dissociation (HCD) recently made it possible to detect the low mass isobaric tag reagent fragments on linear ion trap instruments including the LTQ-Orbitrap (Thermo Scientific) (Meany et al 2007;Armenta, Hoeschele, and Lazar 2009;Kocher et al 2009). The disadvantages of this type of chemical labeling are the presence of potential side reactions, the extra steps required to remove excess reagents and derivatization byproducts resulting in difficulty in achieving complete labeling.…”

Microfluidic chips for protein differential expression profiling

“…Typically, $8-16 mg protein extract injections on bench-top LC-MS instruments resulted in the identification of 300-500 proteins [37]. For exactly the same sample analyzed by bench-top LC-MS/MS, the global iTRAQ ratios were 0.36:1.04:1:4.65 (versus 0.29:0.75:1:3.78 on the chip).…”

“…proteins with po0.001 at a 4twofold change in expression level), putative biomarkers were not found in this experiment, mainly because the biological treatment did not generate differentially expressed proteins among the top $50 proteins that were identified from the injection of $1 mg sample on the chip. However, studies that involved the analysis of larger sample amounts (8-16 mg) with conventional LC-MS enabled the selection of 16 differentially expressed proteins in the E2/Tam-treated cells [37]. One of them, was identified on the chip, as well, i.e.…”