Quantitative site-specific analysis of protein glycosylation by LC-MS using different glycopeptide-enrichment strategies

Wohlgemuth, Jessica; Karas, Michael; Eichhorn, T.; Hendriks, Robertus; Andrecht, Sven

doi:10.1016/j.ab.2009.08.023

Cited by 109 publications

(119 citation statements)

References 53 publications

Supporting

Mentioning

117

Contrasting

Order By: Relevance

“…Glycosylation is usually heterogeneous and a single peptide peak splits into multiple signals, accompanied by reduced signal intensity and altered retention on separation systems when compared with its unglycosylated counterpart (35). Furthermore, glycopeptide signals can be suppressed in the presence of peptides as a result of competitive ionization (45)(46)(47). Therefore, reduction of the sample complexity by a selective enrichment of glycopeptides is often beneficial.…”

mentioning

confidence: 99%

“…Therefore, reduction of the sample complexity by a selective enrichment of glycopeptides is often beneficial. This can be realized by means of chemical capturing techniques (48) or by a solid phase extraction prior, or after a proteolytic digestion (34,46,49). In the case in which single proteins or less complex protein mixtures are investigated, an enrichment can be compensated by a chromatographic separation as it is usually the case in LC-ESI-MS (35).…”

mentioning

confidence: 99%

See 1 more Smart Citation

A Microarray-Matrix-assisted Laser Desorption/Ionization-Mass Spectrometry Approach for Site-specific Protein N-glycosylation Analysis, as Demonstrated for Human Serum Immunoglobulin M (IgM)*

Pabst

Kuster

Wahl³

et al. 2015

Molecular & Cellular Proteomics

View full text Add to dashboard Cite

We demonstrate a new approach for the site-specific identification and characterization of protein N-glycosylation. It is based on a nano-liquid chromatography microarray-matrix assisted laser desorption/ionization-MS platform, which employs droplet microfluidics for onplate nanoliter reactions. A chromatographic separation of a proteolytic digest is deposited at a high frequency on the microarray. In this way, a short separation run is archived into thousands of nanoliter reaction cavities, and chromatographic peaks are spread over multiple array spots. After fractionation, each other spot is treated with PNGaseF to generate two correlated traces within one run, one with treated spots where glycans are enzymatically released from the peptides, and one containing the intact glycopeptides. Mining for distinct glycosites is performed by searching for the predicted deglycosylated peptides in the treated trace. An identified peptide then leads directly to the position of the "intact" glycopeptide clusters, which are located in the adjacent spots. Furthermore, the deglycosylated peptide can be sequenced efficiently in a simple collision-induced dissociation-MS experiment. We applied the microarray approach to a detailed site-specific glycosylation analysis of human serum IgM. By scanning the treated spots with low-resolution matrix assisted laser desorption/ionization-time-offlight-MS, we observed all five deglycosylated peptides, including the one originating from the secretory chain. A detailed glycopeptide characterization was then accomplished on the adjacent, untreated spots with high mass resolution and high mass accuracy using a matrix assisted laser desorption ionization-Fourier transform-MS. We present the first detailed and comprehensive mass spectrometric analysis on the glycopeptide level for human polyclonal IgM with high mass accuracy. Besides complex type glycans on Asn 395, 332, 171, and on the J chain, we observed oligomannosidic glycans on Asn 563, Asn 402 and minor amounts of oligomannosidic glycans on the glycosite Asn 171. Furthermore, hybrid type glycans were found on Asn 402, Asn 171 and in traces Asn 332. Molecular & Cellular

show abstract

mentioning

confidence: 99%

mentioning

confidence: 99%

A Microarray-Matrix-assisted Laser Desorption/Ionization-Mass Spectrometry Approach for Site-specific Protein N-glycosylation Analysis, as Demonstrated for Human Serum Immunoglobulin M (IgM)*

Pabst

Kuster

Wahl³

et al. 2015

Molecular & Cellular Proteomics

View full text Add to dashboard Cite

show abstract

“…To confirm the predicted N-glycosylation sites, the digestion experiment was repeated on the glycosylated protein using α-chymotrypsin to obtain low molecular mass glycopeptides. Despite improvements in mass spectrometric methodologies, analysis of protein glycosylations is still very challenging, because the signals of glycopeptides are often suppressed in the presence of other peptides (Wohlgemuth et al 2009;Geyer and Geyer 2006). For this reason, HILIC enrichments of glycopeptides were performed prior to RP-HPLC/nESI-MS/MS analysis, as described in the experiential section.…”

Section: Resultsmentioning

confidence: 99%

Sequence characterization and glycosylation sites identification of donkey milk lactoferrin by multiple enzyme digestions and mass spectrometry

et al. 2016

View full text Add to dashboard Cite

Lactoferrin, a protein showing an array of biochemical properties, including immuno-modulation, iron-binding ability, as well as antioxidant, antibacterial and antiviral activities, but which may also represent a potential milk allergen, was isolated from donkey milk by ion exchange chromatography. The characterization of its primary structure, by means of enzymatic digestions, SPITC derivatization of tryptic digest, reversed-phase high performance liquid chromatography, electrospray and matrix-assisted laser desorption/ionization mass spectrometry, is reported. Our results allowed the almost complete characterization of donkey lactoferrin sequence, that, at least for the covered sequence, differs from the horse genomic deduced sequence (UniProtKB Acc. Nr. O77811) by five point substitutions located at positions 91 (Arg → His), 328 (Thr → Ile/Leu), 466 (Ala → Gly), 642 (Asn → Ser) and 668 (Ser → Ala). Analysis of the glycosylated protein showed that glycans in donkey lactoferrin are linked to the protein backbone via an amide bond to asparagine residues located at the positions 137, 281 and 476.

show abstract

“…16 Wohlgemuth et al compared silica, cellulose, NH2, amide, and zwitterionic sulfoalkylbetaine (ZIC-HILIC) materials for glycopeptide enrichment using model glycoprotein mixtures prior to reversed-phase capillary LC-MS/MS analysis, and found ZIC-HILIC to be the preferred choice. 41 GCB is an option for the SPE of carbohydrates in water. Historically, the usefulness of GCB was recognized based on the ability of the activated carbon to extract carbohydrates from water.…”

Section: Specific Entrapment Of Glycansmentioning

confidence: 99%

Recent Developments in Liquid Chromatography and Capillary Electrophoresis for the Analysis of Glycoprotein Glycans

Suzuki

2013

ANAL. SCI.

View full text Add to dashboard Cite

Quantitative site-specific analysis of protein glycosylation by LC-MS using different glycopeptide-enrichment strategies

Cited by 109 publications

References 53 publications

A Microarray-Matrix-assisted Laser Desorption/Ionization-Mass Spectrometry Approach for Site-specific Protein N-glycosylation Analysis, as Demonstrated for Human Serum Immunoglobulin M (IgM)*

A Microarray-Matrix-assisted Laser Desorption/Ionization-Mass Spectrometry Approach for Site-specific Protein N-glycosylation Analysis, as Demonstrated for Human Serum Immunoglobulin M (IgM)*

Sequence characterization and glycosylation sites identification of donkey milk lactoferrin by multiple enzyme digestions and mass spectrometry

Recent Developments in Liquid Chromatography and Capillary Electrophoresis for the Analysis of Glycoprotein Glycans

Contact Info

Product

Resources

About