Deeptee Seebun

Large-scale characterization of intact N-glycopeptides using an automated glycoproteomic method

UNLABELLED The detailed characterization of site-specific glycosylation requires the identification of glycan composition and specific attachment sites on proteins, which need the identification of intact glycopeptides by mass spectrometry. In this study, we present an analytical and computational strategy for the high throughput characterization of intact N-glycopeptides derived from complex proteome samples. N-glycopeptides were identified using the spectra acquired for intact glycopeptides as well as de-glycopeptides. The Y1 ion (peptide+GlcNAc) was accurately determined from the spectra of intact glycopeptides, and the structure of glycan was then identified by searching a constructed glycan database with calculated molecular weight of glycans and their fragment ions. The peptide sequences of intact glycopeptides were identified by matching the molecular weight calculated from Y1 ion to that of deglycosylated peptides from the same HILIC enrichment and identified by a separated LC-MS/MS analysis. The fully automated software platform integrates all of the above processes involved in the identification of the intact N-glycopeptides. This platform was applied to detailed characterization of site-specific glycosylation in HEK 293T cells, which led to the identification of 2249 unique intact N-glycopeptides. These intact glycopeptides revealed 1769 site-specific N-glycans on 453 glycosylation sites which demonstrated the high heterogeneity of glycosylations. BIOLOGICAL SIGNIFICANCE We presented a fully automated software platform for the high throughput characterization of intact N-glycopeptides derived from complex proteome samples. Intact glycopeptides and their deglycosylated forms were identified respectively and combined according to the commonality of molecular weights of peptide backbones. The strong correlation of retention times effectively filtered out random matches. The reliability of this strategy was carefully evaluated which showed a probability of random matches less than 1%. In total, 2249 intact glycopeptides were identified which is by the far the largest dataset among the studies of N-glycoproteomics.

Lyso‐form fragment ions facilitate the determination of stereospecificity of diacyl glycerophospholipids

In this work we report the development of a novel methodology for the determination of stereospecificity of diacyl glycerophospholipids, including glycerophosphatidic acids (PA), glycerophosphoserines (PS), glycerophosphoglycerols (PG), glycerophosphoinositols (PI), and glycerophosphoethanolamines (PE), which can be conventionally ionized in negative ion mode. This methodology uses MS(2) recorded on a hybrid quadrupole time-of-flight mass spectrometer to determine the stereospecificity of diacyl glycerophospholipids based on the lyso-form fragment ions, attributed to the neutral loss of fatty acyl moieties. The fragmentation patterns of a variety of diacyl glycerophospholipid standards were first fully examined over a wide range of collision energy. We observed that lyso-form fragment ions corresponding to the neutral loss of fatty acyl moieties attached to the sn2 position as free fatty acids ([M-Sn2](-) ) and as ketenes ([M-(Sn2-H(2) O)](-) ) exhibited consistently higher intensity than their counterpart ions due to the neutral loss of fatty acyl moieties attached to the sn1 position ([M-Sn1](-) and [M-(Sn1-H(2) O)](-) ). Therefore, we concluded that an empirical fragmentation rule can be used to precisely determine the stereospecificity of diacyl glycerophospholipids, primarily on the basis of relative abundance of the lyso-form fragment ions. We then examined the product ion spectra of diacyl glycerophospholipids recorded from lipid extracts of rat hepatoma cells, where the stereospecific information of these lipids was conclusively determined. Combining the novel methodology reported in this work with the currently widely practiced mass spectrometric techniques such as multiple precursor ion scans (MPIS), fatty acyl scans (FAS), and multidimensional mass spectrometry based shotgun lipidomics (MDMS-SL), should enable a reliable and convenient platform for comprehensive glycerophospholipid profiling.

Proteomic reactors and their applications in biology.

Proteomic analysis requires the combination of an extensive suite of technologies including protein processing and separation, micro‐flow HPLC, MS and bioinformatics. Although proteomic technologies are still in flux, approaches that bypass gel electrophoresis (gel‐free approaches) are dominating the field of proteomics. Along with the development of gel‐free proteomics, came the development of devices for the processing of proteomic samples termed proteomic reactors. These microfluidic devices provide rapid, robust and efficient pre‐MS sample procession by performing protein sample preparation/concentration, digestion and peptide fractionation. The proteomic reactor has advanced in two major directions: immobilized enzyme reactor and ion exchange‐based proteomic reactor. This review summarizes the technical developments and biological applications of the proteomic reactor over the last decade.

From cells to peptides: "one-stop" integrated proteomic processing using amphipols.

In proteomics, detergents and chaotropes are indispensable for proteome analysis, not only for protein extraction, but also for protein digestion. To increase the protein extraction efficiency, detergents are usually added in the lysis buffer to extract membrane proteins out of membrane structure and to maintain protein in solutions. In general, these detergents need to be removed prior to protein digestion, usually by precipitation or ultrafiltration. Digestion often takes place in the presence of chaotropic reagents, such as urea, which often need to be removed prior to mass spectrometry. The addition and removal of detergents and chaotropes require multiple steps that are time-consuming and can cause sample losses. Amphipols (APols) are a different class of detergents that have physical and solubilization properties that are distinct from conventional detergents. They have primarily been used in protein structure analysis for membrane protein trapping and stabilization. Here, we demonstrate a simple and rapid protocol for total and membrane proteome preparation using APols. We demonstrate that APols added for cell lysis help maintain the proteome in solution, are compatible with protein digestion using trypsin, and can readily be removed prior to mass spectrometry by a one-step acidification and centrifugation. This protocol is much faster, can be performed in a single tube, and can readily replace the conventional detergent/chaotrope approaches for total and membrane proteome analysis.

Site-specific characterization of cell membrane N-glycosylation with integrated hydrophilic interaction chromatography solid phase extraction and LC–MS/MS

UNLABELLED Glycosylation of membrane proteins plays an important role in cellular behaviors such as cell-cell interaction, immunologic recognition and cell signaling. However, the effective extraction of membrane proteins, the selective isolation of glycopeptides and the mass spectrometric characterization of glycosylation are challenging with current analytical techniques. In this study, a systematic approach was developed which combined: an integrated hydrophilic interaction chromatography solid phase interaction (HILIC SPE) for simultaneous detergent removal and glycopeptide enrichment, and mass spectrometric identification of both protein N-glycosylation sites and site-specific glycan composition. The HILIC SPE conditions were optimized to enable the use of a high concentration of strong detergents, such as SDS and Triton X-100 and to dissolve highly hydrophobic membrane proteins, thus increasing the yield of membrane protein extraction. We illustrated the performance of this approach for the study of membrane protein glycosylation from human embryonic kidney cell lines (HEK 293T). 200μg total protein digest was processed using this approach, leading to the identification of 811 N-glycosylation sites from 567 proteins within two experimental replicates. Furthermore, 177 glycopeptides representing 82 N-glycosites with both glycan composition and peptide sequence were identified by high energy collision dissociation. BIOLOGICAL SIGNIFICANCE A method for systematic characterizing of cell membrane glycosylation has been developed in this manuscript. It is comprised of an integrated hydrophilic interaction chromatography solid phase extraction for the simultaneous detergent removal and intact glycopeptide enrichment. This HILIC SPE significantly increased the efficiency and sensitivity for glycosylation analysis and was combined with high energy collision dissociation to characterize site-specific N-glycosylation from HEK293 cell membrane. Totally 811 N-glycosylation sites from 567 proteins were identified and 177 intact glycopeptides with both glycan composition and peptides sequence were characterized, which provided a solution for site-specific N-glycosylation characterization of membrane.

APols-aided protein precipitation: a rapid method for concentrating proteins for proteomic analysis.

Amphipols (APols) are a newly designed and milder class of detergent. They have been used primarily in protein structure analysis for membrane protein trapping and stabilization. We have recently demonstrated that APols can be used as an alternative detergent for proteome extraction and digestion, to achieve a “One-stop” single-tube workflow for proteomics. In this workflow, APols are removed by precipitation after protein digestion without depleting the digested peptides. Here, we took further advantage of this precipitation characteristic of APols to concentrate proteins from diluted samples. In contrast with tryptic peptides, a decrease in pH leads to the unbiased co-precipitation of APols with proteins, including globular hydrophilic proteins. We demonstrated that this precipitation is a combined effect of acid precipitation and the APols’ protein interactions. Also, we have been able to demonstrate that APols-aided protein precipitation works well on diluted samples, such as secretome sample, and provides a rapid method for protein concentration.

Detecting protein-protein interactions/complex components using mass spectrometry coupled techniques.

Proteins play important roles in biochemical processes. Most biological functions are realized through protein-protein interactions (PPI). Co-immunoprecipitation is the most straightforward method to detect PPI. With the development of modern mass spectrometry (MS), throughput, sensitivity, and confidence for the detection of PPI can be readily achieved by scaling up traditional antibody-based strategies. Herein, we describe a typical workflow for general PPI detection using mass spectrometry coupled techniques, covering from Co-immunoprecipitation (Co-IP), to gel display, in-gel digestion, liquid chromatography mass spectrometry (LC-MS) analysis, as well as result interpretation and statistic filtering. This protocol provides an overview of the technique as well as practical tips.

Associations Between Soluble LDLR and Lipoproteins in a White Cohort and the Effect of PCSK9 Loss-of-Function

Context Elevated circulating cholesterol-rich low-density lipoprotein (LDL) particles increase coronary artery disease risk. Cell-surface hepatic LDL receptors (LDLRs) clear 70% of these particles from circulation. The ectodomain of LDLR is shed into circulation, preventing it from removing LDL particles. The role that LDLR ectodomain shedding plays as a regulatory mechanism is unknown. Objective We describe LDLR shedding via the relationships between circulating soluble LDLRs (sLDLRs) and serum lipoproteins, serum proprotein convertase subtilin/kexin type 9 (PCSK9; a negative regulator of LDLR), and clinical parameters in a white Canadian population. Design Population-based, cross-sectional study. Settings Clinical Research Center, The Ottawa Hospital, and Faculty of Medicine, University of Ottawa. Participants Two hundred seventy-three white Canadians. Intervention None. Main Outcome Measures sLDLR measured by ELISA; serum lipids and PCSK9, PCSK9 genotypes, and clinical parameters from previous analyses. Results sLDLRs correlated strongly with triglycerides (TG; r = 0.624, P < 0.0001) and moderately with LDL cholesterol (r = 0.384, P < 0.0001), and high-density lipoprotein cholesterol (r = -0.307, P = 0.0003). Only TG correlations were unaffected by PCSK9 variations. sLDLR levels were significantly elevated in those with TG >50th or LDL cholesterol >75th percentiles. Conclusions Serum sLDLR levels correlate with several lipoprotein parameters, especially TG, and the presence of PCSK9 loss-of-function variants alters sLDLR levels and correlations, except for TG. Ectodomain LDLR shedding has a role in LDL metabolism, distinct from PCSK9, with interplay between these two pathways that regulate cell-surface LDLRs. Findings suggest alteration of LDLR shedding could emerge as a target to treat dyslipidemia.