A Fully Automated System with Online Sample Loading, Isotope Dimethyl Labeling and Multidimensional Separation for High-Throughput Quantitative Proteome Analysis

Abstract

Multidimensional separation is often applied for large-scale qualitative and quantitative proteome analysis. A fully automated system with integration of a reversed phase-strong cation exchange (RP-SCX) biphasic trap column into vented sample injection system was developed to realize online sample loading, isotope dimethyl labeling and online multidimensional separation of the proteome samples. Comparing to conventionally manual isotope labeling and off-line fractionation technologies, this system is fully automated and time-saving, which is benefit for improving the quantification reproducibility and accuracy. As phosphate SCX monolith was integrated into the biphasic trap column, high sample injection flow rate and high-resolution stepwise fractionation could be easily achieved. Approximately 1000 proteins could be quantified in approximately 30 h proteome analysis, and the proteome coverage of quantitative analysis can be further greatly improved by prolong the multidimensional separation time. This system was applied to analyze the different protein expression level of HCC and normal human liver tissues. After three times replicated analysis, finally 94 up-regulated and 249 down-regulated (HCC/Normal) proteins were successfully obtained. These significantly regulated proteins are widely validated by both gene and proteins expression studies previously. Such as some enzymes involved in urea cycle, methylation cycle and fatty acids catabolism in liver were all observed down-regulated.

In-depth research of multidrug resistance related cell surface glycoproteome in gastric cancer

“…The reconstituted non-glycopeptides in 0.1% formic acid (FA) aqueous solution were analyzed by a 2D nano LC-MS/MS system as described by Wang et al with minor change [19]. The strong cation exchange (SCX) column was connected to an RP C18 column (12 cm × 75 μm i.d) in tandem by a union.…”

A large-scale protein phosphorylation analysis reveals novel phosphorylation motifs and phosphoregulatory networks in Arabidopsis

“…Particularly in the case of capillary LC-MS/MS, online MudPIT has proven to be one of the best solutions for analysis of low abundant biological samples and usually <1 mg of starting protein materials is sufficient [20,23]. In addition, by equipping online MudPIT with novel monolithic columns that have prominent feature of permeability, peak separation capacity in peptides fractionation can be further improved [23][24][25].…”

Recent advances on multidimensional liquid chromatography–mass spectrometry for proteomics: From qualitative to quantitative analysis—A review

“…Wang and coworkers [117] developed a similar fully automatic platform integrating online sample loading, isotope dimethyl labeling and online 2D LC-MS/MS separations, as shown in Fig. 4.…”

“…∼1000 proteins could be quantified in ∼30 h. This system was applied to analyze the differential expression of proteins of Hepatocellular Carcinoma (HCC) and normal human liver tissues, by which 94 up-regulated and 249 down-regulated (HCC/Normal) proteins were successfully obtained after triplicate analysis, and these results were in good Reprinted with permission, from Ref. [117]. Copyright (2010) American Chemical Society.…”