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Plasma Glycopeptide Enrichment

ABSTRACT: We have compared three different glycopeptide enrichment techniques (SAX-ERLIC, HILIC, and lectin affinity) and a C18 control to a tryptic digest of proteins from depleted human plasma.

INSTRUMENT(S): LTQ Orbitrap Elite

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Blood Plasma

SUBMITTER: Sharon Pitteri

LAB HEAD: Sharon Pitteri

PROVIDER: PXD005655 | Pride | 2017-02-16

REPOSITORIES: Pride

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Dataset's files

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			Action	DRS
	C18_Elite_Run1.byparms	Other
	C18_Elite_Run1.byrslt	Other
	C18_Elite_Run1.raw	Raw
	C18_Elite_Run1.xlsx	Xlsx
	C18_Elite_Run2.byparms	Other

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Publications

Parallel Comparison of N-Linked Glycopeptide Enrichment Techniques Reveals Extensive Glycoproteomic Analysis of Plasma Enabled by SAX-ERLIC.

Totten Sarah M SM Feasley Christa L CL Bermudez Abel A Pitteri Sharon J SJ

Journal of proteome research 20170215 3

Protein glycosylation is of increasing interest due to its important roles in protein function and aberrant expression with disease. Characterizing protein glycosylation remains analytically challenging due to its low abundance, ion suppression issues, and microheterogeneity at glycosylation sites, especially in complex samples such as human plasma. In this study, the utility of three common N-linked glycopeptide enrichment techniques is compared using human plasma. By analysis on an LTQ-Orbitra ...[more]

PMID: 28199111

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Finding the Sweet Spot in ERLIC Mobile Phase for Simultaneous Enrichment of N-Glyco and Phosphopeptides

Project description:Simultaneous enrichment of glyco- and phospho- peptides will benefit the studies of biological processes regulated by these post-translational modifications (PTMs). It will also reveal potential crosstalk between these two ubiquitous PTMs. Unlike custom designed multifunctional solid phase extraction (SPE) materials, operating strong-anion exchange (SAX) resin in electrostatic repulsion-hydrophilic interaction chromatography (ERLIC) mode provides a readily available strategy to analytical labs for enrichment of these PTMs for subsequent mass spectrometry (MS)-based characterization. However, the choice of mobile phase has largely relied on empirical rules from hydrophilic interaction chromatography (HILIC) or ion-exchange chromatography (IEX) without further optimization and adjustments. In this study, ten mobile phase compositions of ERLIC were systematically compared; the impact of multiple factors including organic phase proportion, ion pairing reagent, pH and salt on the retention of glycosylated and phosphorylated peptides were evaluated. This study demonstrated good enrichment of glyco- and phospho- peptides from the nonmodified peptides in a complex tryptic digest. Moreover, the enriched glyco- and phospho- peptides elute in different fractions by orthogonal retention mechanisms of hydrophilic interaction and electrostatic interaction in ERLIC, maximizing the LC-MS identification of each PTM. The optimized mobile phase can be adapted to the ERLIC HPLC system, where the high resolution in separating multiple PTMs will benefit large-scale MS-based PTM profiling and in-depth characterization.

2019-09-06 | PXD015179 | Pride

Intact Glycopeptide Analysis of Human Urine

Project description:Identification of intact glycopeptides in pooled healthy human urine samples

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Anion-Exchange Chromatography of Tryptic and Phosphopeptides: WAX vs. SAX and AEX vs. ERLIC

Project description:At a pH > 5, phosphopeptides have two negative charges per residue and are well-retained in anion-exchange chromatography. However, the peptides with one or two phosphate groups are not separated from the peptides with multiple Asp or Glu residues, which interfere with the identification of phosphopeptides in tryptic digests. At a pH around 2, phosphate residues have just a single negative charge but Asp and Glu have none. This facilitates the separation of phosphopeptides from unmodified acidic peptides. The singly phosphorylated peptides are retained too weakly under these conditions, due to electrostatic repulsion, unless hydrophilic interaction is superimposed in the ERLIC mode (electrostatic repulsion-hydrophilic interaction chromatography). Weak anion-exchange (WAX) and strong anion-exchange (SAX) columns were compared, both with peptide standards and with a complex tryptic digest. The SAX column exhibited higher capacity at pH 6 than did the WAX column. However, only about 60% as many phosphopeptides were identified at pH 6 than via ERLIC at pH 2. In one run, 12,467 phosphopeptides were identified, including 4233 with more than one phosphate. We conclude that chromatography of phosphopeptides is best performed at low pH in the ERLIC mode. Under those conditions the performances of the SAX and WAX materials were comparable.

2015-04-23 | PXD001333 | Pride

Proteome-wide discovery of human brain protein glycoforms and glycan modification changes in Alzheimer’s disease

Project description:We performed a large-scale, intact glycopeptide-based glycoproteomics study of human brain tissue samples from neuropathologically confirmed AD cases and their age-matched controls. The study provided a system-level view of human brain protein glycoforms and site-specific glycan modifications. Our analyses revealed previously unknown changes in protein glycoforms and site-specific glycans in AD brain. Our findings provide novel insights into the roles of glycan modifications in brain dysfunction in AD.

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Counterion Optimization Dramatically Improves Selectivity for Phosphopeptides and Glycopeptides in Electrostatic Repulsion-Hydrophilic Interaction Chromatography (ERLIC)

Project description:A well-hydrated counterion can selectively and dramatically increase retention of a charged analyte in hydrophilic interaction chromatography (HILIC). The effect is enhanced if the column is charged, as in electrostatic repulsion-hydrophilic interaction chromatography (ERLIC). This combination was exploited in proteomics for the isolation of peptides with certain post-translational modifications (PTMs). The best salt additive examined was magnesium trifluoroacetate. The well-hydrated Mg+2 ion promoted retention of peptides with functional groups that retained negative charge at low pH, while the poorly-hydrated trifluoroacetate counterion tuned down the retention due to the basic residues. The result was an enhancement in selectivity between 6- to 66-fold. These conditions were applied to a tryptic digest of mouse cortex. Gradient elution produced fractions enriched in peptides with phosphate, mannose-6-phosphate, N- and O-linked glycans. The numbers of such peptides identified either equaled or exceeded the numbers afforded by the best alternative methods. This method is a productive and convenient way to isolate peptides simultaneously that contain a number of different PTMs, facilitating study of proteins with “crosstalk” modifications. The fractions from the ERLIC column were desalted prior to C-18-reversed phase (RP) LC-MS/MS analysis. Between 47-100% of the peptides with more than one phosphate or sialyl- residue or with a mannose-6 phosphate group were not retained by a C-18 cartridge but were retained by a cartridge of porous graphitic carbon. This finding implies that the abundance of such peptides may have been significantly underestimated in some past studies.

2021-06-01 | PXD022988 | Pride

ERLIC-based strategies for combined quantitative proteome and phosphoproteome analysis with limited sample amounts

Project description:The study aims the development of a sensitive strategy for combined quantitative proteomics and phosphoproteomics. 3 ERLIC-based strategies, applicable for low-sample-amount experiments (~150 µg per condition), were evaluated and compared . Three samle sets have been uploaded. 16 fractions ERLIC-1-SCX/RP (2 replicates) for phosphopeptide enrichment, 8 fractions SCX-EL (2 replicates), 12 fractions ERLIC-2 (2 replicates). The following 3 strategies were compared: A 2D strategy -> sample set ERLIC-1-SCX/RP. A pseudo-3Dstrategy -> sample sets ERLIC-1-SCX/RP + SCX-EL and a 3D strategy -> sample sets ERLIC-1-SCX/RP + ERLIC-2

2015-02-25 | PXD001241 | Pride

HILIC and ERLIC Enrichment of Glycopeptides Derived from Breast and Brain Cancer Cells

Project description:Aberrant glycosylation has been linked to many different cancer types. The blood brain barrier (BBB) is a region of the brain that regulates the entrance of ions, diseases, toxins, etc. However in breast cancer metastasis, the BBB fails to prevent the crossing of the cancer cells into the brain. Here we present a study of identifying and quantifying the glycosylation of six breast and brain cancer cell lines using hydrophilic interaction liquid chromatography (HILIC) and electrostatic repulsion liquid chromatography (ERLIC) enrichments and LC-MS/MS analysis. Qualitative and quantitative analyses of N-linked glycosylation were performed by both enrichment techniques for individual and complementary comparison. Potential cancer glycopeptide biomarkers were identified and confirmed by chemometric and statistical evaluations. The results provide the first comprehensive glycopeptide listing for these six cell lines.

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Optimal Dissociation Methods Differ for N- and O-glycopeptides

Project description:Site-specific characterization of glycosylation requires intact glycopeptide analysis, and recent efforts have focused on how to best interrogate glycopeptides using tandem mass spectrometry (MS/MS). Beam-type collisional activation, i.e., higher-energy collisional dissociation (HCD), has been a valuable approach, but stepped collision energy HCD (sceHCD) and electron transfer dissociation with HCD supplemental activation (EThcD) have emerged as potentially more suitable alternatives. Both sceHCD and EThcD have been used with success in large-scale glycoproteomic experiments, but they each incur some degree of compromise. Furthermore, N-glycoproteomics has made significant progress in the last few years, and there is growing interest in extending this progress to O-glycoproteomics, which necessitates comparisons of method performance for the two classes of glycopeptides. Here, we systematically explore the advantages and disadvantages of conventional HCD, sceHCD, ETD, and EThcD for intact glycopeptide analysis and comment on their suitability for both N- and O-glycoproteomic applications. For N-glycopeptides, HCD and sceHCD generate similar numbers of identifications, although sceHCD generally provides higher quality spectra. Both significantly outperform EThcD methods, indicating that ETD-based methods are not required for routine N-glycoproteomics. Conversely, ETD-based methods, especially EThcD, are indispensable for site-specific analyses of O-glycopeptides. Our data show that O-glycopeptides cannot be robustly characterized with HCD-centric methods that are sufficient for N-glycopeptides, and glycoproteomic methods aiming to characterize O-glycopeptides must be constructed accordingly.

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