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Description
| - Determining disease-associated changes in protein glycosylation provides a better understanding of pathogenesis. This work focuses on human immunoglobulin A1 (IgA1), where aberrant O-glycosylation plays a key role in the pathogenesis of IgA nephropathy (IgAN). Normal IgA1 hinge region carries 3 to 6 O-glycans consisting of N-acetylgalactosamine (GalNAc) and galactose (Gal); both sugars may be sialylated. In IgAN patients, some O-glycans on a fraction of IgA1 molecules are Gal-deficient. Here we describe a sample preparation protocol with optimized cysteine alkylation of a Gal-deficient polymeric IgA1 myeloma protein prior to in-gel digestion and analysis of the digest by MALDI-TOF/TOF mass spectrometry (MS). Following a novel strategy, IgA1 hinge-region O-glycopeptides were fractionated by reversed-phase liquid chromatography using a microgradient device and identified by MALDI-TOF/TOF tandem MS (MS/MS). The acquired MS/MS spectra were interpreted manually and by means of our own software. This allowed assigning up to six O-glycosylation sites and demonstration, for the first time, of the distribution of isomeric O-glycoforms having the same molecular mass, but a different glycosylation pattern. The most abundant Gal-deficient O-glycoforrns were GalNAc(4)Gal(3) and GalNAc(5)Gal(4) with one Gal-deficient site and GalNAc(5)Gal(3) and GalNAc(4)Gal(2) with two Gal-deficient sites. The most frequent Gal-deficient sites were at Ser230 and/or Thr236.
- Determining disease-associated changes in protein glycosylation provides a better understanding of pathogenesis. This work focuses on human immunoglobulin A1 (IgA1), where aberrant O-glycosylation plays a key role in the pathogenesis of IgA nephropathy (IgAN). Normal IgA1 hinge region carries 3 to 6 O-glycans consisting of N-acetylgalactosamine (GalNAc) and galactose (Gal); both sugars may be sialylated. In IgAN patients, some O-glycans on a fraction of IgA1 molecules are Gal-deficient. Here we describe a sample preparation protocol with optimized cysteine alkylation of a Gal-deficient polymeric IgA1 myeloma protein prior to in-gel digestion and analysis of the digest by MALDI-TOF/TOF mass spectrometry (MS). Following a novel strategy, IgA1 hinge-region O-glycopeptides were fractionated by reversed-phase liquid chromatography using a microgradient device and identified by MALDI-TOF/TOF tandem MS (MS/MS). The acquired MS/MS spectra were interpreted manually and by means of our own software. This allowed assigning up to six O-glycosylation sites and demonstration, for the first time, of the distribution of isomeric O-glycoforms having the same molecular mass, but a different glycosylation pattern. The most abundant Gal-deficient O-glycoforrns were GalNAc(4)Gal(3) and GalNAc(5)Gal(4) with one Gal-deficient site and GalNAc(5)Gal(3) and GalNAc(4)Gal(2) with two Gal-deficient sites. The most frequent Gal-deficient sites were at Ser230 and/or Thr236. (en)
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Title
| - Elucidating heterogeneity of IgA1 hinge-region O-glycosylation by use of MALDI-TOF/TOF mass spectrometry: Role of cysteine alkylation during sample processing
- Elucidating heterogeneity of IgA1 hinge-region O-glycosylation by use of MALDI-TOF/TOF mass spectrometry: Role of cysteine alkylation during sample processing (en)
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skos:prefLabel
| - Elucidating heterogeneity of IgA1 hinge-region O-glycosylation by use of MALDI-TOF/TOF mass spectrometry: Role of cysteine alkylation during sample processing
- Elucidating heterogeneity of IgA1 hinge-region O-glycosylation by use of MALDI-TOF/TOF mass spectrometry: Role of cysteine alkylation during sample processing (en)
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skos:notation
| - RIV/60162694:G44__/13:43875042!RIV14-MO0-G44_____
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http://linked.open...avai/riv/aktivita
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http://linked.open...avai/riv/aktivity
| - I, P(ED0007/01/01), P(EE2.4.31.0130)
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http://linked.open...iv/cisloPeriodika
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http://linked.open...vai/riv/dodaniDat
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http://linked.open...aciTvurceVysledku
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http://linked.open.../riv/druhVysledku
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http://linked.open...iv/duvernostUdaju
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http://linked.open...titaPredkladatele
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http://linked.open...dnocenehoVysledku
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http://linked.open...ai/riv/idVysledku
| - RIV/60162694:G44__/13:43875042
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http://linked.open...riv/jazykVysledku
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http://linked.open.../riv/klicovaSlova
| - Microgradient separation; Mass spectrometry; Glycopeptide; O-glycosylation; IgA nephropathy; Human immunoglobulin A1 (IgA1) (en)
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http://linked.open.../riv/klicoveSlovo
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http://linked.open...odStatuVydavatele
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http://linked.open...ontrolniKodProRIV
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http://linked.open...i/riv/nazevZdroje
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http://linked.open...in/vavai/riv/obor
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http://linked.open...ichTvurcuVysledku
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http://linked.open...cetTvurcuVysledku
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http://linked.open...vavai/riv/projekt
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http://linked.open...UplatneniVysledku
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http://linked.open...v/svazekPeriodika
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http://linked.open...iv/tvurceVysledku
| - Novák, Jan
- Stulík, Jiří
- Řehulka, Pavel
- Franc, Vojtěch
- Raus, Martin
- Renfrow, Matthew B.
- Sebela, Marek
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http://linked.open...ain/vavai/riv/wos
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issn
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number of pages
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http://bibframe.org/vocab/doi
| - 10.1016/j.jprot.2013.07.013
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http://localhost/t...ganizacniJednotka
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