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. 2013 Jan 15;85(2):956-63.
doi: 10.1021/ac302574f. Epub 2012 Dec 28.

In-gel Nonspecific Proteolysis for Elucidating Glycoproteins: A Method for Targeted Protein-Specific Glycosylation Analysis in Complex Protein Mixtures

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Free PMC article

In-gel Nonspecific Proteolysis for Elucidating Glycoproteins: A Method for Targeted Protein-Specific Glycosylation Analysis in Complex Protein Mixtures

Charles C Nwosu et al. Anal Chem. .
Free PMC article

Abstract

Determining protein-specific glycosylation in protein mixtures remains a difficult task. A common approach is to use gel electrophoresis to isolate the protein followed by glycan release from the identified band. However, gel bands are often composed of several proteins. Hence, release of glycans from specific bands often yields products not from a single protein but a composite. As an alternative, we present an approach whereby glycans are released with peptide tags allowing verification of glycans bound to specific proteins. We term the process in-gel nonspecific proteolysis for elucidating glycoproteins (INPEG). INPEG combines rapid gel separation of a protein mixture with in-gel nonspecific proteolysis of protein bands followed by tandem mass spectrometry (MS) analysis of the resulting N- and O-glycopeptides. Here, in-gel digestion is shown for the first time with nonspecific and broad specific proteases such as Pronase, proteinase K, pepsin, papain, and subtilisin. Tandem MS analysis of the resulting glycopeptides separated on a porous graphitized carbon (PGC) chip was achieved via nanoflow liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (nano-LC/Q-TOF MS). In this study, rapid and automated glycopeptide assignment was achieved via an in-house software (Glycopeptide Finder) based on a combination of accurate mass measurement, tandem MS data, and predetermined protein identification (obtained via routine shotgun analysis). INPEG is here initially validated for O-glycosylation (κ casein) and N-glycosylation (ribonuclease B). Applications of INPEG were further demonstrated for the rapid determination of detailed site-specific glycosylation of lactoferrin and transferrin following gel separation and INPEG analysis on crude bovine milk and human serum, respectively.

Figures

Figure 1
Figure 1
1-D gel analysis of (A) various amounts of a mixture of kappa casein and ribonuclease B, (B) bovine milk proteins, and (C) human serum proteins.
Figure 2
Figure 2
(A) Extracted compound chromatogram (ECC) of kappa casein glycopeptides generated following pronase digestion of 10 μg of the protein. (B) Detailed site-heterogeneity and detected glycoform abundance of kappa casein following INPEG analysis.
Figure 3
Figure 3
(A) Extracted compound chromatogram (ECC) of ribonuclease B glycopeptides generated following pronase digestion of 10 μg of the protein. (B) Detailed site-heterogeneity and detected glycoform abundance of ribonuclease B following INPEG analysis.
Figure 4
Figure 4
Comparison of glycopeptide abundances following INPEG analysis of 10 μg, 1 μg, 100 ng and 10 ng of gel-loaded ribonuclease B.
Figure 5
Figure 5
(A) Extracted compound chromatogram (ECC) of glycopeptides generated from INPEG analysis of a bovine milk gel band containing bovine lactoferrin, polymeric immunoglobulin receptor and serotransferrin. (B) Detailed site-heterogeneity and detected glycoform abundance of bovine lactoferrin following INPEG analysis.
Figure 6
Figure 6
(A) Extracted compound chromatogram (ECC) of glycopeptides gener ated from INPEG analysis of a human serum gel band containing human transferring and immunoglobulin M. (B) Detailed site-heterogeneity and detected glycoform abundance of human transferrin.

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