Detection of glycoproteins separated by nondenaturing polyacrylamide gel electrophoresis using the periodic acid-Schiff stain

Doerner, Kinchel C.; White, Bryan A.

doi:10.1016/0003-2697(90)90433-a

Cited by 89 publications

(47 citation statements)

References 7 publications

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“…Enzymes were incubated with BSM in a 1:175 (wt/wt) ratio for 20 h and with fetuin or ASF in a 1:25 (wt/wt) ratio for 3 h, in 20 mM Tris·HCl pH 7.5 at 37°C. The samples were separated on 10-15% SDS/PAGE and stained for specific glycoprotein detection with the periodic acid-Schiff (PAS) stain (30). Briefly, gels were fixed in 50% (vol/vol) methanol for 30 min, washed with 5% (vol/vol) acetic acid (3 × 10 min), and incubated with 0.7% (wt/vol) periodic acid in 5% acetic acid solution for 3 h at room temperature (RT).…”

Section: Methodsmentioning

confidence: 99%

Recognition of protein-linked glycans as a determinant of peptidase activity

Noach

Ficko-Blean

Pluvinage

et al. 2017

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

137

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The vast majority of proteins are posttranslationally altered, with the addition of covalently linked sugars (glycosylation) being one of the most abundant modifications. However, despite the hydrolysis of protein peptide bonds by peptidases being a process essential to all life on Earth, the fundamental details of how peptidases accommodate posttranslational modifications, including glycosylation, has not been addressed. Through biochemical analyses and X-ray crystallographic structures we show that to hydrolyze their substrates, three structurally related metallopeptidases require the specific recognition of O-linked glycan modifications via carbohydrate-specific subsites immediately adjacent to their peptidase catalytic machinery. The three peptidases showed selectivity for different glycans, revealing protein-specific adaptations to particular glycan modifications, yet always cleaved the peptide bond immediately preceding the glycosylated residue. This insight builds upon the paradigm of how peptidases recognize substrates and provides a molecular understanding of glycoprotein degradation.

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Section: Methodsmentioning

confidence: 99%

Recognition of protein-linked glycans as a determinant of peptidase activity

Noach

Ficko-Blean

Pluvinage

et al. 2017

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

137

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“…Pili were purified from the M. maripaludis ⌬flaK mutant, a nonflagellated but piliated mutant of strain S2, using the protocol described previously (72). Protein samples were stained with the PAS reagent to detect glycoproteins (23). A whole-cell lysate of M. marisnigri, which has a glycoprotein S layer of 138 kDa known to be stained by PAS (77), was included on the same gel as an internal control.…”

Section: Vol 193 2011 Pili Of Methanococcus Maripaludis 805mentioning

confidence: 99%

Genetic and Mass Spectrometry Analyses of the Unusual Type IV-Like Pili of the ArchaeonMethanococcus maripaludis

Nair

et al. 2011

J Bacteriol

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The structure of pili from the archaeon Methanococcus maripaludis is unlike that of any bacterial pili. However, genetic analysis of the genes involved in the formation of these pili has been lacking until this study. Pili were isolated from a nonflagellated (⌬flaK) mutant and shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to consist primarily of subunits with an apparent molecular mass of 17 kDa. In-frame deletions were created in three genes, MMP0233, MMP0236, and MMP0237, which encode proteins with bacterial type IV pilin-like signal peptides previously identified by in silico methodology as likely candidates for pilus structural proteins. Deletion of MMP0236 or MMP0237 resulted in mutant cells completely devoid of pili on the cell surface, while deletion of the third pilin-like gene, MMP0233, resulted in cells greatly reduced in the number of pili on the surface. Complementation with the deleted gene in each case returned the cells to a piliated state. Surprisingly, mass spectrometry analysis of purified pili identified the major structural pilin as another type IV pilin-like protein, MMP1685, whose gene is located outside the first pilus locus. This protein was found to be glycosylated with an N-linked branched pentasaccharide glycan. Deletion and complementation analysis confirmed that MMP1685 is required for piliation.

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“…Other modifications made to the PAS staining method have included the capacity for quick detection [205] and applicability to proteins resolved by native PAGE [206]. Alternative applications of the PAS stain for detection of glycoproteins include combining the PAS reaction with alcian blue or dansyl chloride staining [207][208][209][210].…”

Section: Glycosylated Proteinsmentioning

confidence: 99%

Quantitative proteomics: assessing the spectrum of in-gel protein detection methods

2010

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Proteomics research relies heavily on visualization methods for detection of proteins separated by polyacrylamide gel electrophoresis. Commonly used staining approaches involve colorimetric dyes such as Coomassie Brilliant Blue, fluorescent dyes including Sypro Ruby, newly developed reactive fluorophores, as well as a plethora of others. The most desired characteristic in selecting one stain over another is sensitivity, but this is far from the only important parameter. This review evaluates protein detection methods in terms of their quantitative attributes, including limit of detection (i.e., sensitivity), linear dynamic range, inter-protein variability, capacity for spot detection after 2D gel electrophoresis, and compatibility with subsequent mass spectrometric analyses. Unfortunately, many of these quantitative criteria are not routinely or consistently addressed by most of the studies published to date. We would urge more rigorous routine characterization of stains and detection methodologies as a critical approach to systematically improving these critically important tools for quantitative proteomics. In addition, substantial improvements in detection technology, particularly over the last decade or so, emphasize the need to consider renewed characterization of existing stains; the quantitative stains we need, or at least the chemistries required for their future development, may well already exist.

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Detection of glycoproteins separated by nondenaturing polyacrylamide gel electrophoresis using the periodic acid-Schiff stain

Cited by 89 publications

References 7 publications

Recognition of protein-linked glycans as a determinant of peptidase activity

Recognition of protein-linked glycans as a determinant of peptidase activity

Genetic and Mass Spectrometry Analyses of the Unusual Type IV-Like Pili of the ArchaeonMethanococcus maripaludis

Quantitative proteomics: assessing the spectrum of in-gel protein detection methods

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