Site-specific characterization and quantitation ofN-glycopeptides in PKM2 knockout breast cancer cells using DiLeu isobaric tags enabled by electron-transfer/higher-energy collision dissociation (EThcD)

Abstract

The system-wide site-specific analysis of intact glycopeptides is crucial for understanding the exact functional relevance of protein glycosylation. A dedicated workflow with the capability to simultaneously characterize and quantify intact glycopeptides in a site-specific and high-throughput manner is essential to reveal specific glycosylation alteration patterns in complex biological systems. In this study, an enhanced, dedicated, large-scale site-specific quantitative N-glycoproteomics workflow has been established, which includes improved specific extraction of membrane-bound glycoproteins using the filter aided sample preparation (FASP) method, enhanced enrichment of N-glycopeptides using sequential hydrophilic interaction liquid chromatography (HILIC) and multi-lectin affinity (MLA) enrichment, site-specific N-glycopeptide characterization enabled by EThcD, relative quantitation utilizing isobaric N,N-dimethyl leucine (DiLeu) tags and automated FDR-based large-scale data analysis by Byonic. For the first time, our study shows that HILIC complements to a very large extent to MLA enrichment with only 20% overlapping in enriching intact N-glycopeptides. When applying the developed workflow to site-specific N-glycoproteome study in PANC1 cells, we were able to identify 1067 intact N-glycopeptides, representing 311 glycosylation sites and 88 glycan compositions from 205 glycoproteins. We further applied this approach to study the glycosylation alterations in PKM2 knockout cells vs. parental breast cancer cells and revealed altered N-glycoprotein/N-glycopeptide patterns and very different glycosylation microheterogeneity for different types of glycans. To obtain a more comprehensive map of glycoprotein alterations, N-glycopeptides after treatment with PNGase F were also analyzed. A total of 484 deglycosylated peptides were quantified, among which 81 deglycosylated peptides from 70 glycoproteins showed significant changes. KEGG pathway analysis revealed that the PI3K/Akt signaling pathway was highly enriched, which provided evidence to support the previous finding that PKM2 knockdown cancer cells rely on activation of Akt for their survival. With glycosylation being one of the most important signaling modulators, our results provide additional evidence that signaling pathways are closely regulated by metabolism.

Glycoproteomic markers of hepatocellular carcinoma‐mass spectrometry based approaches

“…However, the ability to analyze the glycopeptides has remained a challenge. In recent work, there have been a number of techniques developed to deal with this problem (Table ), including collision‐induced dissociation (CID)/electron‐transfer dissociation (ETD) (Alley et al, ), CID/higher‐energy collisional dissociation (HCD) (Segu & Mechref, ; Lee et al, ), stepped HCD (Liu et al, ; Yin et al, ), and electron‐transfer/higher‐energy collision dissociation (EThcD) (Yu et al, ; Chen et al, ; Glover et al, ), for example. The use of CID/ETD MS to analyze glycopeptides has been recently reviewed by Mechref (Mechref, ).…”

Quantitative site- and structure-specific N-glycoproteomics characterization of differential N-glycosylation in MCF-7/ADR cancer stem cells

“…At least two ratios need to be observed among the three technical replicates. For the intact N-glycopeptides quantitated at least twice, the p value was calculated using t-test [19]; and the intact N-glycopeptides with a fold change of no less than 1.5 and p value no bigger than 0.05 were classified as differentially expressed intact N-glycopeptides.…”

Human protein paucimannosylation: cues from the eukaryotic kingdoms

“…Recent glycomics and glycoproteomics‐centric studies have demonstrated that PMPs are also elevated glycosylation signatures associated with various human cancers including those of ovarian (Everest‐Dass et al ., ; Chen et al ., ), colorectal (Joosten et al ., ; Balog et al ., ; Sethi et al ., ; Kaprio et al ., ; Holst et al ., ), breast (Lee et al ., ; Chen et al ., ), lung (Hua et al ., ; Ruhaak et al ., ; Wang et al ., ), paraganglioma (Leijon et al ., ), glioblastoma multiforme (Becker et al ., ), skin (Moginger et al ., ) and prostate (Shah et al ., ) origin. By systematically interrogating a very large collection of nearly 500 N ‐glycomics LC‐MS/MS data sets obtained from 11 cancer types and subtypes, we have obtained solid evidence that PMGs are significant features of human cancers and, at least in part, expressed on cancer cell surfaces (S. Chatterjee, L. Y. Lee, R. Kawahara, J. L. Abrahams, B. Adamczyk, M. Anugraham, C. Ashwood, Z. Sumer‐Bayraktar, M. T. Briggs, J. H. L. Chik, A. Everest‐Dass, S. Förster, H. Hinneburg, K. R. M. Leite, I. Loke, U. Möginger, E. S. X. Moh, M. Nakano, S. Recuero, M. K. Sethi, M. Srougi, K. Stavenhagen, V. Venkatakrishnan, K. Wongtrakul‐Kish, S. Diestel, P. Hoffmann, N. G. Karlsson, D. Kolarich, M. P. Molloy, M. H. Muders, M. K. Oehler, N. H. Packer, G. Palmisano & M. Thaysen‐Andersen, in preparation).…”