Recent Advances in Glycoproteomic Analysis by Mass Spectrometry

Suttapitugsakul, Suttipong; Sun, Fangxu; Wu, Ronghu

doi:10.1021/acs.analchem.9b04651

Cited by 120 publications

(85 citation statements)

References 346 publications

(561 reference statements)

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“…Next to the diversity originating from distinct genotypes and processing, further variety in Hp stems from posttranslational modifications (PTMs), primarily glycosylation ( 32 – 34 ). Protein glycosylation is one of the most complex PTMs and dramatically influences the structural and functional properties of proteins.…”

mentioning

confidence: 99%

A wealth of genotype-specific proteoforms fine-tunes hemoglobin scavenging by haptoglobin

Tamara

Franc

Heck

2020

Proc. Natl. Acad. Sci. U.S.A.

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The serum haptoglobin protein (Hp) scavenges toxic hemoglobin (Hb) leaked into the bloodstream from erythrocytes. In humans, there are two frequently occurring allelic forms of Hp, resulting in three genotypes: Homozygous Hp 1-1 and Hp 2-2, and heterozygous Hp 2-1. The Hp genetic polymorphism has an intriguing effect on the quaternary structure of Hp. The simplest form, Hp 1-1, forms dimers consisting of two α1β units, connected by disulfide bridges. Hp 2-1 forms mixtures of linear (α1)2(α2)n-2(β)noligomers (n> 1) while Hp 2-2 occurs in cyclic (α2)n(β)noligomers (n> 2). Different Hp genotypes bind Hb with different affinities, with Hp 2-2 being the weakest binder. This behavior has a significant influence on Hp’s antioxidant capacity, with potentially distinctive personalized clinical consequences. Although Hp has been studied extensively in the past, the finest molecular details of the observed differences in interactions between Hp and Hb are not yet fully understood. Here, we determined the full proteoform profiles and proteoform assemblies of all three most common genetic Hp variants. We combined several state-of-the-art analytical methods, including various forms of chromatography, mass photometry, and different tiers of mass spectrometry, to reveal how the tens to hundreds distinct proteoforms and their assemblies influence Hp’s capacity for Hb binding. We extend the current knowledge by showing that Hb binding does not just depend on the donor’s genotype, but is also affected by variations in Hp oligomerization, glycosylation, and proteolytic processing of the Hp α-chain.

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confidence: 99%

A wealth of genotype-specific proteoforms fine-tunes hemoglobin scavenging by haptoglobin

Tamara

Franc

Heck

2020

Proc. Natl. Acad. Sci. U.S.A.

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“…Currently, common methods for sample preparation of tryptic N-glycopeptide enrichment using HILIC or other materials usually obtain relatively few high-confidence intact N-glycopeptide species [ 21 , 22 ]. Different protease combinations have also been used in proteomic research [ 5 ]; however, this strategy was rarely applied to investigate intact N-glycopeptides.…”

Section: Resultsmentioning

confidence: 99%

In-depth characterization and comparison of the N-glycosylated proteome of two-dimensional- and three-dimensional-cultured breast cancer cells and xenografted tumors

et al. 2020

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Native intact N-glycopeptide analysis can provide access to the comprehensive characteristics of N-glycan occupancy, including N-glycosites, N-glycan compositions, and N-glycoproteins for complex samples. The sample pre-processing method used for the analysis of intact N-glycopeptides usually depends on the enrichment of low abundance N-glycopeptides from a tryptic peptide mixture using hydrophilic substances before LC-MS/MS detection. However, the number of identified intact N-glycopeptides remains inadequate to achieve an in-depth profile of the N-glycosylation landscape. Here, we optimized the sample preparation workflow prior to LC-MS/MS analysis by systematically comparing different analytical methods, including the use of different sources of trypsin, combinations of different proteases, and different enrichment materials. Finally, we found that the combination of Trypsin (B)/Lys-C digestion and zwitterionic HILIC (Zic-HILIC) enrichment significantly improved the mass spectrometric characterization of intact N-glycopeptides, increasing the number of identified intact N-glycopeptides and offering better analytical reproducibility. Furthermore, the optimized workflow was applied to the analysis of intact N-glycopeptides in two-dimensional (2D) and three-dimensional (3D)-cultured breast cancer cells in vitro and xenografted tumors in mice. These results indicated that the same breast cancer cells, when cultured in different microenvironments, can show different N-glycosylation patterns. This study also provides an interesting comparison of the N-glycoproteome of breast cancer cells cultured in different growth conditions, indicating the important role of N-glycosylated proteins in cancer cell growth and the choice of the cell culture model for studies in tumor biology and drug evaluation.

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“…The workflow describes processing steps in the O-Pair Search strategy, which generates a fragment ion index [1,2] and O-glycan groups [3,4] from user defined protein and O-glycan databases, respectively. Using an ultrafast, fragment-index-enabled open modification search [5] paired with a match of delta masses to aggregate glycan mass combinations [6] enables identification of O-glycopeptide candidates from HCD spectra [7].…”

Section: Figure 1 O-pair Search Through Metamorpheus For Fast and Comentioning

confidence: 99%

O-Pair Search with MetaMorpheus for O-glycopeptide Characterization

Lü

Riley

Shortreed

et al. 2020

Preprint

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We report O-Pair Search, a new approach to identify O-glycopeptides and localize O-glycosites. Using paired collision-and electron-based dissociation spectra, O-Pair Search identifies Oglycopeptides using an ion-indexed open modification search and localizes O-glycosites using graph theory and probability-based localization. O-Pair Search reduces search times more than 2,000-fold compared to current O-glycopeptide processing software, while defining O-glycosite localization confidence levels and generating more O-glycopeptide identifications. O-Pair Search is freely available: https://github.com/smith-chem-wisc/MetaMorpheus. Main TextMass spectrometry (MS) is the gold standard for interrogating the glycoproteome, enabling the localization of glycans to specific glycosites. 1-3 Recent applications of electron-driven dissociation methods have shown promise in localizing modified O-glycosites even in multiply glycosylated peptides 4 . Yet, standard approaches for interpreting tandem MS spectra are ill-suited for the heterogeneity of O-glycopeptides. Perhaps the most challenging problem for Oglycopeptide analysis is mucin-type O-glycosylation, which is abundant on many extracellular and secreted proteins and is a crucial mediator of immune function, microbiome interaction, and biophysical forces imposed on cells, among others 5 . Mucin-type O-glycans are linked to serine and threonine residues through an initiating N-acetylgalactosamine (GalNAc) sugar, which can be further elaborated into four major core structures (cores 1-4) or remain truncated as terminal GalNAc (Tn) and sialyl-Tn antigens 6 . These O-glycosites occur most frequently in long serine/threonine rich sequences (Supplementary Fig. 1), such as PTS mucin tandem repeat domains, which exist with microheterogeneity defined by a large number of potential O-glycans 7 . The number of serine and threonine residues present in glycopeptides derived from mucin-type O-glycoproteins, combined with the consideration of dozens of potential O-glycans at each site, leads to a combinatorial explosion when generating databases of theoretical O-glycopeptides to consider for each tandem MS/MS spectrum (Supplementary Note 1). AUTHOR INFORMATION Affiliations

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Recent Advances in Glycoproteomic Analysis by Mass Spectrometry

Cited by 120 publications

References 346 publications

A wealth of genotype-specific proteoforms fine-tunes hemoglobin scavenging by haptoglobin

A wealth of genotype-specific proteoforms fine-tunes hemoglobin scavenging by haptoglobin

In-depth characterization and comparison of the N-glycosylated proteome of two-dimensional- and three-dimensional-cultured breast cancer cells and xenografted tumors

O-Pair Search with MetaMorpheus for O-glycopeptide Characterization

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