Analysis of protein C termini is very important for functional annotations of proteomes, while proteome-wide C termini analysis still poses substantial challenges. Here we described a simple and robust strategy for specific isolation of protein C termini based on LysC digestion and site-selective dimethylation to deplete N-terminal and internal peptides by scavenger materials. The performance of LysC digestion and conditions of site-selective dimethylation and resin coupling were discussed in detail. Then the strategy was successfully applied to the characterization of protein C termini of HeLa cells. A total of 781 protein C termini were identified with a 300 μg digest in our study, among which 38.9% were actually not identifiable using current trypsin digestion-based methods due to their inappropriate peptide length for MS analysis, indicating that our method was highly complementary to the existing methods. The enrichment procedure was rapid and easy to operate and could afford a very good identification efficiency by obtaining the largest C termini data set of the human proteome with the least sample loading. This method was without bias toward physicochemical properties of peptides. Moreover, a peptide-centric database was first introduced to analyze protein C termini, which effectively improved the accuracy and speed of the database search. Therefore, our method can be used to effectively and selectively isolate protein C termini and contributes to the global annotation of C terminomes.

Quantitative N-glycomics can reveal abnormal expression of N-glycan in diseases. However, mass spectrometry (MS)-based N-glycome quantitative analysis is still technically challenging. To achieve the quantitation of Nglycome with high accuracy and sensitivity, it is required to efficiently label the N-glycans with isotopic tags and selectively enrich N-glycans to avoid suppression from other substances. Herein, we developed an integrated pipeline that combines isotopically fluorous tag labeling and fluorous solid-phase extraction to quantitatively analyze the N-glycome by MS. In this strategy, the N-glycans were labeled with light and heavy aminoxy-functionalized fluorous tags (PFBHA and PFBHAd 2 ) through the oxime click reaction. Then through the fluorous solid-phase extraction, the fluorous tag labeled N-glycan could be purified from contaminants like salts and proteins for the following quantitative analysis by mass spectrometry. This new approach enables selective purification (molar ratio of glycan to protein at 1:100) and accurate (R 2 > 0.99) and reproducible (coefficient of variation (CV)) < 25%, n = 6) quantitation of Nglycans within 2 orders of magnitude. Uniquely, diagnostic ions (D and [D-221]) were generated in tandem MS analysis after fluorous tags labeling, which could be used to deduce the composition of the 6-antenna and to distinguish isomers. Finally, this strategy was successfully applied to analyze the N-glycan changes in human serum associated with hepatocellular carcinoma (HCC). Fifteen N-glycan compositions with bisecting GlcNAc, sialic acid, and core fucosylation showed significant differences in HCC serum.

Protein N-terminal acetylation (Nα-acetylation) is one of the most common modifications in both eukaryotes and prokaryotes. Although studies have shown that Nα-acetylation plays important roles in protein assembly, stability, and location, the physiological role has not been fully elucidated. Therefore, a robust and large-scale analytical method is important for a better understanding of Nα-acetylation. Here, an enrichment strategy was presented based on LysN digestion and amine-reactive resin capture to study naturally acetylated protein N termini. Since LysN protease cleaves at the amino-terminus of the lysine residue, all resulting peptides except naturally acetylated N-terminal peptides contain free amino groups and can be removed by coupling with AminoLink Resin. Therefore, the naturally acetylated N-terminal peptides were left in solution and enriched for further liquid chromatography–tandem mass spectrometry (LC–MS/MS) analysis. The method was very simple and fast, which contained no additional chemical derivatization except protein reduction and alkylation necessarily needed in bottom-up proteomics. It could be used to study acetylated N termini from complex biological samples without bias toward different peptides with various physicochemical properties. The enrichment specificity was above 99% when it was applied in HeLa cell lysates. Neo-N termini generated by endogenous degradation could be directly distinguished without the use of stable-isotope labeling because no chemical derivatization was introduced in this method. Furthermore, this method was highly complementary to the traditional analytical methods for protein N termini based on trypsin only with ArgC-like activity. Therefore, the described method was beneficial to naturally acetylated protein N termini profiling.