Shan Yichu

Label-free quantification of differentially expressed proteins in mouse liver cancer cells with high and low metastasis rates by a SWATH acquisition method

Label-free quantification is a valuable tool for the analysis of differentially expressed proteins identified by mass spectrometry methods. Herein, we used a new strategy: data-dependent acquisition mode identification combined with label-free quantification by SWATH acquisition mode, to study the differentially expressed proteins in mouse liver cancer metastasis cells. A total of 1528 protein groups were identified, among which 1159 protein groups were quantified and 249 protein groups were observed as differentially expressed proteins (86 proteins up-regulated and 163 down-regulated). This method provides a commendable solution for the identification and quantification of differentially expressed proteins in biological samples.

Enrichment strategy of protein N-termini based on dioctyl labeling

The analysis of protein N-termini provides valuable information for protein structure and function annotation, and helps the profiling of proteases substrates and cleavage sites. However, most of the current N-terminal enrichment approaches involve multiple chromatographic separation processes and require a large amount of scavenger materials, which make it time and labor consuming and may induce significant sample loss. Herein, we develop a negative N-termini enrichment strategy based on dioctyl labeling. With yeast lysate digests as the sample, the strategy showed a high efficiency in dioctyl labeling and retention shift. Such a strategy was applied for the enrichment of N-terminal peptides from yeast cell lysates and enabled the identification of 237 original N-termini and 133 neo-N-termini, which was improved by 100% and 480% compared with direct analysis. Furthermore, with the combination of multi-proteases digestion, the number of protein N-termini and neo-N-termin was improved by 50% and 90%, respectively, facilitating the in-depth N-terminome analysis.

Novel Algorithm for Identification and Quantification of Proteins Based on Strategy of Isobaric Peptide Termini Labelling

Isobaric peptide termini labeling (IPTL) is a technology which uses light and heavy isotopes to label C-terminus and N-terminus of peptides. As the masses of labeled peptides are equivalent, the complexity of sample is low when analyzing MS data produced by this technology. Besides, paired b and y ions are helpful while analyzing MS/MS data in this kind of experiments. On the basis of this, a novel scoring algorithm, all ions scoring algorithm (AISA), has been designed for IPTL experiments. The information of quantification and qualification can be acquired at the same time using AISA. On Q-Exactive HeLa 2D RPLC dataset, peptide spectrum matches (PSMs), distinct peptides and protein groups identified by AISA are 15%, 26% and 22% higher than Morpheus. On human-HCC-HL dataset, PSMs, distinct peptides and protein groups identified by AISA are 24%, 39% and 27% higher than Morpheus. Quantification ratio on Q-Exactive HeLa and human. HCC-HL datasets are 1.18 and 0.90, respectively, which are very close to 1. Besides, quantification ratios between 0.5 and 2.0 are 91% and 94%, respectively.