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Elucidation of O-glycosylation structures of the beta-amyloid precursor protein by liquid chromatography-mass spectrometry using electron transfer dissociation and collision induced dissociation.

ABSTRACT: Accumulation and deposition of beta-amyloid peptide, a major constituent in neuritic plaques are hallmarks of Alzheimer's disease (AD) and AD-related neurodegenerative diseases. beta-Amyloid (Abeta) is derived from the proteolytic cleavage of amyloid precursor protein (APP), a transmembrane protein present in three major isoforms in brain comprising 695, 751 and 770 amino acids, respectively. Among other post-translational modifications, APP is modified during maturation by N- and O-glycosylation, which are thought to be responsible for its expression and secretion. Unlike N-glycosylation, no sites of O-glycosylation of APP have previously been reported. We report here the identification of three specific O-glycosylation sites of the secreted APP695 (sAPP695) produced in CHO cells, using a combination of high-performance liquid chromatography and electrospray-tandem mass spectrometry. With the use of electron transfer dissociation and collision induced dissociation (ETD and CID), we identified type, composition and structures of the Core 1 type O-linked glycans attached at the residues Thr 291, Thr 292 and Thr 576 of the full-length APP695. The glycosylations comprise multiple short glycans, containing N-acetyl galactosamine (GalNAc), Gal-GalNAc and sialic acid terminated structures. The presence of the glycopeptides in the tryptic mixture was identified using the CID-generated sugar oxonium ions. ETD proved to be valuable for the unambiguous identification of the modified sites as ETD fragmentation occurred along the peptide backbone with little or no cleavage of the glycans. Thus, the combination of the CID and ETD techniques in LC-MS is shown here, as a powerful tool for de novo identification of O-glycosylations at unknown modification sites in proteins.

SUBMITTER: Perdivara I

PROVIDER: S-EPMC2743936 | biostudies-literature | 2009 Feb

REPOSITORIES: biostudies-literature

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Elucidation of O-glycosylation structures of the beta-amyloid precursor protein by liquid chromatography-mass spectrometry using electron transfer dissociation and collision induced dissociation.

Perdivara Irina I Petrovich Robert R Allinquant Bernadette B Deterding Leesa J LJ Tomer Kenneth B KB Przybylski Michael M

Journal of proteome research 20090201 2

Accumulation and deposition of beta-amyloid peptide, a major constituent in neuritic plaques are hallmarks of Alzheimer's disease (AD) and AD-related neurodegenerative diseases. beta-Amyloid (Abeta) is derived from the proteolytic cleavage of amyloid precursor protein (APP), a transmembrane protein present in three major isoforms in brain comprising 695, 751 and 770 amino acids, respectively. Among other post-translational modifications, APP is modified during maturation by N- and O-glycosylatio ...[more]

PMID: 19093876

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Project description:Site-specific analysis of protein glycosylation is important for biochemical and clinical research efforts. Glycopeptide analysis using liquid chromatography-collision-induced dissociation/electron transfer dissociation mass spectrometry (LC-CID/ETD-MS) allows simultaneous characterization of the glycan structure and attached peptide site. However, due to the low ionization efficiency of glycopeptides during electrospray ionization, 200-500 fmol of sample per injection is needed for a single LC-MS run, which makes it challenging for the analysis of limited amounts of glycoprotein purified from biological matrixes. To improve the sensitivity of LC-MS analysis for glycopeptides, an ultranarrow porous layer open tubular (PLOT) LC column (2.5 m × 10 μm i.d.) was coupled to a linear ion trap (LTQ) collision-induced dissociation/electron transfer dissociation mass spectrometer to provide sensitive analysis of N-linked protein glycosylation heterogeneity. The potential of the developed method is demonstrated by the characterization of site-specific glycosylation using haptoglobin (Hpt) as a model protein. To limit the amount of haptoglobin to low picomole amounts of protein, we affinity purified it from 1 μL of pooled lung cancer patient plasma. A total of 26 glycoforms/glycan compositions on three Hpt tryptic glycopeptides were identified and quantified from 10 LC-MS runs with a consumption of 100 fmol of Hpt digest (13 ng of protein, 10 fmol per injection). Included in this analysis was the determination of the glycan occupancy level. At this sample consumption level, the high sensitivity of the PLOT LC-LTQ-CID/ETD-MS system allowed glycopeptide identification and structure determination, along with relative quantitation of glycans presented on the same peptide backbone, even for low abundant glycopeptides at the ∼100 amol level. The PLOT LC-MS system is shown to have sufficient sensitivity to allow characterization of site-specific protein glycosylation from trace levels of glycosylated proteins.

Project description:Glycopeptide-level mass spectrometry (MS) and tandem mass spectrometry (MS/MS) analyses are commonly performed to establish site-specific protein glycosylation profiles that are of central importance to gaining structure-function insights on glycoproteins. Confoundingly, the complete characterization of glycopeptide connectivity usually requires the acquisition of multiple MS/MS fragmentation spectra. Complementary ion fragmentation techniques such as collision-induced dissociation (CID) and electron transfer dissociation (ETD) are often applied in concert to address this need. While structurally informative, the requirement for acquisition of two MS/MS spectra per analyte places considerable limitations upon the breadth and depth of large-scale glycoproteomic inquiry. Here, a previously developed method of multiplexing CID and ETD is applied to the study of glycopeptides for the first time. Integration of the two dissociation methods was accomplished through addition of an ion mobility (IM) dimension that disperses the two stages of MS/MS in time. This allows the two MS/MS spectra to be acquired within a few milliseconds of one another, and to be deconvoluted in post-processing. Furthermore, the method allows both fragmentation readouts to be obtained from the same precursor ion packet, thus reducing the inefficiencies imposed by separate CID and ETD acquisitions and the relatively poor precursor ion to fragment ion conversion typical of ETD. N-Linked glycopeptide ions ranging in molecular weight from 1.8 to 6.5 kDa were generated from four model glycoproteins that collectively encompassed paucimannosidic, high mannose, and complex types of N-glycosylation. In each case, IM-resolved CID and ETD events provided complete coverage of the glycan topology and peptide sequence coverages ranging from 48.4% (over 32 amino acid residues) to 85.7% (over eight amino acid residues). The potential of this method for large-scale glycoproteomic analysis is discussed.

Project description:We present a workflow using an ETD-optimised version of Mascot Percolator and a modified version of SLoMo (turbo-SLoMo) for analysis of phosphoproteomic data. We have benchmarked this against several database searching algorithms and phosphorylation site localisation tools and show that it offers highly sensitive and confident phosphopeptide identification and site assignment with PSM-level statistics, enabling rigorous comparison of data acquisition methods. We analysed the Plasmodium falciparum schizont phosphoproteome using for the first time, a data-dependent neutral loss-triggered-ETD (DDNL) strategy and a conventional decision-tree method. At a posterior error probability threshold of 0.01, similar numbers of PSMs were identified using both methods with a 73% overlap in phosphopeptide identifications. The false discovery rate associated with spectral pairs where DDNL CID/ETD identified the same phosphopeptide was <1%. 72% of phosphorylation site assignments using turbo-SLoMo without any score filtering, were identical and 99.8% of these cases are associated with a false localisation rate of <5%. We show that DDNL acquisition is a useful approach for phosphoproteomics and results in an increased confidence in phosphopeptide identification without compromising sensitivity or duty cycle. Furthermore, the combination of Mascot Percolator and turbo-SLoMo represents a robust workflow for phosphoproteomic data analysis using CID and ETD fragmentation.Biological significanceProtein phosphorylation is a ubiquitous post-translational modification that regulates protein function. Mass spectrometry-based approaches have revolutionised its analysis on a large-scale but phosphorylation sites are often identified by single phosphopeptides and therefore require more rigorous data analysis to unsure that sites are identified with high confidence for follow-up experiments to investigate their biological significance. The coverage and confidence of phosphoproteomic experiments can be enhanced by the use of multiple complementary fragmentation methods. Here we have benchmarked a data analysis pipeline for analysis of phosphoproteomic data generated using CID and ETD fragmentation and used it to demonstrate the utility of a data-dependent neutral loss triggered ETD fragmentation strategy for high confidence phosphopeptide identification and phosphorylation site localisation.

Dataset Information

Elucidation of O-glycosylation structures of the beta-amyloid precursor protein by liquid chromatography-mass spectrometry using electron transfer dissociation and collision induced dissociation.

Publications

Elucidation of O-glycosylation structures of the beta-amyloid precursor protein by liquid chromatography-mass spectrometry using electron transfer dissociation and collision induced dissociation.

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