Possible target-related proteins and signal network of bufalin in A549 cells suggested by both iTRAQ-based and label-free proteomic analysis.

Bufalin (BF) exhibited antiproliferation and antimigration effects on human A549 lung cancer cells. To search its target-related proteins, protein expression profiles of BF-treated and control cells were compared using two quantitative proteomic methods, iTRAQ-based and label-free proteomic analysis. A total of 5428 proteins were identified in iTRAQ-based analysis while 6632 proteins were identified in label-free analysis. The number of common identified proteins of both methods was 4799 proteins. By application of 1.20-fold for upregulated and 0.83-fold for downregulated cutoff values, 273 and 802 differentially expressed proteins were found in iTRAQ-based and label-free analysis, respectively. The number of common differentially expressed proteins of both methods was 45 proteins. Results of bioinformational analysis using Metacore(TM) showed that the two proteomic methods were complementary and both suggested the involvement of oxidative stress and regulation of gene expression in the effects of BF, and fibronectin-related pathway was suggested to be an important pathway affected by BF. Western blotting assay results confirmed BF-induced change in levels of fibronectin and other related proteins. Overexpression of fibronectin by plasmid transfection ameliorated antimigration effects of BF. Results of the present study provided information about possible target-related proteins and signal network of BF.

ITMSQ: A software tool for N- and C-terminal fragment ion pairs based isobaric tandem mass spectrometry quantification.

Tandem MS (MS2) quantification using the series of N- and C-terminal fragment ion pairs generated from isobaric-labelled peptides was recently considered an accurate strategy in quantitative proteomics. However, the presence of multiplexed terminal fragment ion in MS2 spectra may reduce the efficiency of peptide identification, resulting in lower identification scores or even incorrect assignments. To address this issue, we developed a quantitative software tool, denoted isobaric tandem MS quantification (ITMSQ), to improve N- and C-terminal fragment ion pairs based isobaric MS2 quantification. A spectrum splitting module was designed to separate the MS2 spectra from different samples, increasing the accuracy of both identification and quantification. ITMSQ offers a convenient interface through which parameters can be changed along with the labelling method, and the result files and all of the intermediate files can be exported. We performed an analysis of in vivo terminal amino acid labelling labelled HeLa samples and found that the numbers of quantified proteins and peptides increased by 13.64 and 27.52% after spectrum splitting, respectively. In conclusion, ITMSQ provides an accurate and reliable quantitative solution for N- and C-terminal fragment ion pairs based isobaric MS2 quantitative methods.

Global in vivo terminal amino acid labeling for exploring differential expressed proteins induced by dialyzed serum cultivation.

Taking advantage of reliable metabolic labeling and accurate isobaric MS2 quantification, we developed a global in vivo terminal amino acid labeling (G-IVTAL) strategy by combining metabolic labeling and isotopic dimethyl labeling for quantifying tryptic peptides. With G-IVTAL, the scale of qualitative and quantitative data can be increased twofold compared with in vivo termini amino acid labeling (IVTAL) in which Lys-N and Arg-C are used for digestion. As a result, up to 81.78% of the identified proteins have been confidently quantified in G-IVTAL-labeled HepG2 cells. Dialyzed serum has been used in most SILAC studies to ensure complete labeling. However, dialysis requires the removal of low molecular weight hormones, cytokines, and cellular growth factors, which are essential for the cell growth of certain cell lines. To address the influence of dialyzed serum in HepG2 growth, the G-IVTAL strategy was applied to quantify the expression differences between dialyzed serum- and normal serum-cultured HepG2 cells. Finally, we discovered 111 differentially expressed proteins, which could be used as references to improve the reliability of the SILAC quantification. Among these, by using western blotting, the differential expressions of MTDH, BCAP31, and GPC3 were confirmed as being influenced by dialyzed serum. The experimental results demonstrate that the G-IVTAL strategy is a powerful tool to achieve accurate and reliable protein quantification.

A novel quantitative proteomics workflow by isobaric terminal labeling.

Quantification by series of b, y fragment ion pairs generated from isobaric-labeled peptides in MS2 spectra has recently been considered an accurate strategy in quantitative proteomics. Here we developed a novel MS2 quantification approach named quantitation by isobaric terminal labeling (QITL) by coupling (18)O labeling with dimethylation. Trypsin-digested peptides were labeled with two (16)O or (18)O atoms at their C-termini in H(2)(16)O or H(2)(18)O. After blocking all ε-amino groups of lysines through guanidination, the N-termini of the peptides were accordingly labeled with formaldehyde-d(2) or formaldehyde. These indistinguishable, isobaric-labeled peptides in MS1 spectra produce b, y fragment ion pairs in the whole mass range of MS2 spectra that can be used for quantification. In this study, the feasibility of QITL was first demonstrated using standard proteins. An accurate and reproducible quantification over a wide dynamic range was achieved. Then, complex rat liver samples were used to verify the applicability of QITL for large-scale quantitative analysis. Finally, QITL was applied to profile the quantitative proteome of hepatocellular carcinoma (HCC) and adjacent non-tumor liver tissues. Given its simplicity, low-cost, and accuracy, QITL can be widely applied in biological samples (cell lines, tissues, and body fluids, etc.) for quantitative proteomic research.

In vivo termini amino acid labeling for quantitative proteomics.

Quantitative proteomics is one of the research hotspots in the proteomics field and presently maturing rapidly into an important branch. The two most typical quantitative methods, stable isotope labeling with amino acids in cell culture (SILAC) and isobaric tags for relative and absolute quantification (iTRAQ), have been widely and effectively applied in solving various biological and medical problems. Here, we describe a novel quantitative strategy, termed "IVTAL", for in vivo termini amino acid labeling, which combines some advantages of the two methods above. The core of this strategy is a set of heavy amino acid (13)C(6)-arginine and (13)C(6)-lysine and specific endoproteinase Lys-N and Arg-C that yield some labeled isobaric peptides by cell culture and enzymatic digestion, which are indistinguishable in the MS scan but exhibit multiple MS/MS reporter b, y ion pairs in a full mass range that support quantitation. Relative quantification of cell states can be achieved by calculating the intensity ratio of the corresponding reporter b, y ions in the MS/MS scan. The experimental analysis for various proportions of mixed HeLa cell samples indicated that the novel strategy showed an abundance of reliable quantitative information, a high sensitivity, and a good dynamic range of nearly 2 orders of magnitude. IVTAL, as a highly accurate and reliable quantitative proteomic approach, is expected to be compatible with any cell culture system and to be especially effective for the analysis of multiple post-translational modificational sites in one peptide.