Establishment, Evaluation And Application Of An Integrated Quantitative Proteomic Strategy Based On Acetyl Stable Isotope Labeling And LC-FTICR MS

In recent years, the focus of the proteome research moves towards quantitative rather than solely qualitative analysis. In order to realize the proteome quantitative analysis, many methods have been developed, such as the quantitative strategies based on 2-DE, label-free, mass-coded and stable isotopic labeling. Nowadays, taking the quantitative accuracy and feasibility into account, stable isotopic tagging technique coupled with online liquid chromatography (LC) and mass spectrometry (MS) has been adopted widely. While lots of commercial stable isotope reagents have been provided, there are still some limitations in commercial ones, such as expensive reagent (iTRAQ), few labeling sites (ICAT), dependence on the designated mass spectrometer, and limited availability of quantitative analysis software.The aim of our research is to develop an integrated analysis platform: stable isotopic labeling, on-line LC with MS and automated software, which achieves the high-throughput, high-accuracy and high-sensitivity quantitative proteome analysis. As a result, the novel strategy successfully avoided the limitations of the commercial reagents and overcomed the dependences on the commercial ones.In our research, a d₀/d₃-acetyl stable isotopic labeling quantitative strategy was established and optimized first of all. The tagging sites are the reductive amino of the peptides. There are several major advantages in d₀/d₃-acetyl labeling method, such as tagging all the proteins or peptides, the predicable mass difference between the labeled peptides, simple labeling procedure, mild reactive conditions, good stability of the labeling group in LC, no extra ions in MS, wide compatibility including human sample and cheap reagent. After labeling, each peptide will be incorporated into one acetyl group at least (no less than 3 deuterium atoms). Although the incorporation of higher number of deuterium atoms will decrease the overlap between the labeled peptides, the isotopic effect caused by deuterium atom in reverse phase liquid chromatography (RPLC) will be severe. So, a compatible quantitative strategy is provided: d₀/d₃-acetyl stable isotopic labeling, nano-RPLC, online spotting and matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF/TOF-MS). The quantitative result of the protein mixture demonstrated the feasibility and accuracy of the above strategy in comparative proteome research.In the large-scale proteome research, compared with MALDI MS, electrospray ionization mass spectrometry (ESI-MS) is more comprehensively applied. To make d₀/d₃-acetyl stable isotopic labeling fit for LC-ESI-MS, we exploited another novel quantitative method: guanidination and acetylation stable isotopic labeling (GA labeling). Besides keeping the merits of acetylation labeling, GA labeling excellently controlled the deuterium atoms isotopic effect. Guanidination blockedε-amino and then makes sure each peptide was incorporated with only one acetyl group (3 deuterium atoms), which ensured the same retention times of the labeled peptides. The quantitative analysis of protein mixture indicated that the above strategy has good accuracy, good sensitivity and good reproducibility.Base on GA labeling characteristics and high resolution data of the hybrid linear ion trap fourier-transform ion cyclotron resonance (LTQ-FTICR) MS, a quantitative software 'MSAQ' was developed for automated mass data processing.To evaluate the feasibility of the above integrated analysis platform - GA labeling, FT-MS for data acquisition and a graphic user interface program called 'MSAQ' for automatic data processing, we applied it on three complex biological samples for proteome analysis. Firstly, the whole strategy was applied on the research of proteins N-terminal peptides. Afterε-amino group of lysine residues of the proteins was blocked by guanidination reaction, the resultant proteins were equally divided into two parts and labeled by d₀-acetic anhydride and d₆-acetic anhydride respectively. At last, the labeled proteins were mixed at 1:1, digested by trypsin and analyzed by MS. Only N-terminal peptide of the protein showed a pair of characteristic isotopic peaks with a mass shift of 3.0188 Da, and other peptides were still singlet isotopic peaks. After N-termini of four model proteins were recognized and sequenced, 77 N-terminal peptides from Escherichia coli proteins were also successfully identified. Among them, 46 N-terminal sequences are consistent with database, 31 N-terminal sequences renewed the database annotation, and 9 N-termini whose signal peptides removed were also identified and sequenced. Compared with Edman degradation, our strategy has three advantages: high sensitivity, high specificity and high throughput. Such a result adequately shows the feasibility of the whole strategy in N-termini special recognition and sequencing.Secondly, the whole strategy was applied on the quantitative analysis of CYP450 proteins during the injury of rat hepatic induced by carbon tetrachloride (CCl₄). 17 CYP450 proteins were quantified accurately. Among them, 2E1 was down-regulated dramatically, which was coincident with the previous results. Besides, other CYP450 proteins were also changed significantly. The results indicated that GA labeling not only validates the previous results but also disclosed the alterations of other CYP450 proteins, which would explain the response of these metabolic proteins during the liver injury in a full-scale and systematical angle.Thirdly, the whole strategy was applied on the quantitative analysis of human plasma proteins. Human plasma contains thousands of distinct proteins and its concentration dynamic range is over 12 magnitude orders. For example, 22 most abundant human plasma proteins, such as HSA, IgG, transferring,α2-macroglobulin, andα1-antitrypsin, account for 99% of the protein amount, and then less than 1% protein amount covers more than million proteins.Whether or not the whole strategy can realize the high-throughput, high-sensitivity, high accuracy quantitative analysis of human plasma proteins is a key point of its feasibility in proteomics.At first, 1:1 labeled human plasma proteins was analyzed. The error between the expected value and the experiment value was less than 5%, which illuminated the quantitative accuracy. Afterwards, samples from 5 different phases of human hepatitis plasma were compared and quantified. The 1025 plasma proteins were identified and quantified. Among them, 185 plasma proteins varied evidently, including 20ng/ml heparin cofactor 2 precursor (concentration < 20ng/ml) and angiotensinogen precursor (concentration < 10pg/ml), which proved our method achieves quantitative analysis of 9 magnitude orders. To test the quantitative accuracy, one dramatically different protein, fibronectin, was validated by western-blot. The immunology quantitative result coincided well with the GA labeling result.In summary, we developed the integrated quantitative strategy, which could be used in samples not only from prokaryote and eukaryote, but also from tissue and body fluid. At the same time, our method holds good compatibility either with LC or PAGE separation system. Through the above 3 proteome applications, the feasibility of our technique in high-throughput, high-sensitivity and high-accuracy proteome quantitative research was further verified. So we believe our whole quantitative strategy will own its splendid perspective!...