Sub-micron silica particles coated with polyacrylamide brush layer for protein analysis
Abstract
Protein glycosylation is the most common post-translational modification occurring in living biological systems, which participates in several essential cellular activities. Top-down proteomics has many advantages over the traditional techniques for glycosylation analysis but it requires highly efficient separation techniques for intact glycoproteins as well as other hydrophilic protein variances. The objective of this work is to develop a material based on silica nanoparticles for separation of intact proteins under the hydrophilic interaction liquid chromatography (HILIC) mode. A brush layer of polyacrylamide (PAAm) has been synthesized on sub-micron silica particles. An ultra-thin layer chromatography (UTLC) was made by coating a glass or silicon wafer slide with these PAAm coated particles. A HILIC separation of three proteins followed by on-plate MALDI-MS detection was demonstrated on the UTLC slide. The MALDI analysis of proteins using PAAm coated silica nanoparticles on silicon wafer was further studied with two different laser sources: nitrogen and Nd:YAG/KTP. An ultrahigh pressure liquid chromatography (UHPLC) column was made with sub-micron silica particles coated with PAAm brush layer for HILIC separation. Compared to current leading market HILIC columns, the column with PAAm brush layer has demonstrated better resolution and better selectivity. Two monoclonal antibodies (MAbs) were analyzed using this HILIC column with both PDA and ESI-MS detections and heterogeneity of light chains in the MAbs was discovered.
Degree
Ph.D.
Advisors
Wirth, Purdue University.
Subject Area
Analytical chemistry
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