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Sub 2-?m macroporous silica particles derivatized for enhanced lectin affinity enrichment of glycoproteins.

ABSTRACT: A new, mechanically stable silica microparticle with macrosized internal pores (1.6 ?m particles with 100 nm pores) has been developed for chromatography. The particles are characterized by an extensive network of interconnected macropores with a high intraparticle void volume, as observed by transmission electron microscopy (TEM). They are synthesized by an aerosol assembly technique called ultrasonic spray pyrolysis (USP). The particles have a high surface area for a macroporous material, ?200 m(2)/g, making them suitable for large biomolecular separations. To demonstrate their potential for bioseparations, they have been functionalized with lectins for affinity enrichment of glycoproteins. The material was derivatized with two lectins, Concanavalin A (Con A) and Aleuria aurantia lectin (AAL), and binding properties were tested with standard glycoproteins. The columns exhibited excellent binding capacities for microaffinity enrichment: Con A was able to bind 75 ?g of a standard glycoprotein in a 50 × 1 mm column. Following initial tests, the lectin microcolumns were utilized for enrichment of glycoproteins from 1 ?L volumes of blood serum samples, performed in triplicate for each lectin. The enriched serum fractions were subjected to side-by-side glycomic and glycoproteomic profiling analyses with mass spectrometry to show that the new particles offer excellent sensitivity for microscale analyses of precious biological sample materials. The unique combination of the macroporous architecture and small particle diameter suggests the material may have advantages for conventional modes of chromatographic separation of macromolecules in an ultra-high-pressure liquid chromatography (UHPLC) format.

SUBMITTER: Mann BF

PROVIDER: S-EPMC3586544 | biostudies-literature | 2013 Feb

REPOSITORIES: biostudies-literature

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Sub 2-μm macroporous silica particles derivatized for enhanced lectin affinity enrichment of glycoproteins.

Mann Benjamin F BF Mann Amanda K P AK Skrabalak Sara E SE Novotny Milos V MV

Analytical chemistry 20130117 3

A new, mechanically stable silica microparticle with macrosized internal pores (1.6 μm particles with 100 nm pores) has been developed for chromatography. The particles are characterized by an extensive network of interconnected macropores with a high intraparticle void volume, as observed by transmission electron microscopy (TEM). They are synthesized by an aerosol assembly technique called ultrasonic spray pyrolysis (USP). The particles have a high surface area for a macroporous material, ∼200 ...[more]

PMID: 23278114

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Project description:The glycosylation of proteins is one of the most common post-translational modifications (PTMs) and plays important regulatory functions in diverse biological processes such as protein stability or cell signaling. Accordingly, glycoproteins are also a consistent part of the human tear film proteome, maintaining the proper function of the ocular surface and forming the first defense barrier of the ocular immune system. Irregularities in the glycoproteomic composition of tear film might promote the development of chronic eye diseases, indicating glycoproteins as a valuable source for biomarker discovery or drug target identification. Therefore, the present study aimed to develop a lectin-based affinity method for the enrichment and concentration of tear glycoproteins/glycopeptides and to characterize their specific N-glycosylation sites by high-resolution mass spectrometry (MS). For method development and evaluation, we first accumulated native glycoproteins from human tear sample pools and assessed the enrichment efficiency of different lectin column systems by 1D gel electrophoresis and specific protein stainings (Coomassie and glycoproteins). The best-performing multi-lectin column system (comprising the four lectins ConA, JAC, WGA, and UEA I, termed 4L) was applied to glycopeptide enrichment from human tear sample digests, followed by MS-based detection and localization of their specific N-glycosylation sites. As the main result, our study identified a total of 26 N glycosylation sites of 11 N-glycoproteins in the tear sample pools of healthy individuals (n = 3 biological sample pools). Amongst others, we identified tear film proteins lactotransferrin (N497 and N642, LTF), Ig heavy chain constant α-1 (N144 and 340, IGHA1), prolactin-inducible protein (N105, PIP), and extracellular lacritin (N105, LACRT) as highly reliable and significant N glycoproteins, already associated with the pathogenesis of various chronic eye diseases such as dry eye syndrome (DES). In conclusion, the results of the present study will serve as an important tear film N-glycoprotein catalog for future studies focusing on human tear film and ocular surface-related inflammatory diseases.

Project description:Glycosylation of proteins is one of the most common post-translational modification (PTM) and plays important regulatory functions in diverse biological processes such as protein stability or cell signaling. Accordingly, glycoproteins are also a consistent part of the human tear film proteome maintaining the proper function of the ocular surface and forming the first defense barrier of the ocular immune system. Irregularities in the glycoproteomic composition of the tear film might promote development of chronic eye diseases indicating glycoproteins as valuable source for biomarker discovery or drug target identification. The present study aimed to develop a lectin-based affinity method for the enrichment and concentration of tear glycoproteins/glycopeptides and the characterization of their specific N-glycosylation sites by high-resolution mass spectrometry (MS). For method development and evaluation, we accumulated first native glycoproteins from human tear sample pools and assessed the enrichment efficiency of different lectin column systems by 1D gel electrophoresis and specific protein stainings (Coomassie and glycoproteins). The best-performing multi-lectin column system (comprising the four lectins ConA, JAC, WGA and UEA I, termed as 4L) was applied for glycopeptide enrichment from human tear sample digests followed by MS-based detection and localization of their specific N-glycosylation sites. As main result, the present study identified in total 26 N glycosylation sites of 11 N-glycoproteins in tear sample pools of healthy individuals (n=3 biological sample pools). Amongst others, we identified tear film proteins lactotransferrin (N497 and N642, LTF), Ig heavy chain constant α-1 (N144 and 340, IGHA1), prolactin-inducible protein (N105, PIP) as well as extracellular lacritin (N105, LACRT) as highly reliable and significant N glycoproteins, which were already associated with the pathogenesis of various chronic eye diseases such as the dry eye syndrome (DES). In conclusion, the results of the present study will serve as important tear film N-glycoprotein catalogue for future studies focusing the human tear film and ocular surface-related inflammatory diseases.

Dataset Information

Sub 2-?m macroporous silica particles derivatized for enhanced lectin affinity enrichment of glycoproteins.

Publications

Sub 2-μm macroporous silica particles derivatized for enhanced lectin affinity enrichment of glycoproteins.

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