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Rapid and automatic on‐plate desalting protocol for MALDI‐MS: Using imprinted hydrophobic polymer template
Author(s) -
Jia Weitao,
Wu Huixia,
Lu Haojie,
Li Na,
Zhang Yang,
Cai Ruifang,
Yang Pengyuan
Publication year - 2007
Publication title -
proteomics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.26
H-Index - 167
eISSN - 1615-9861
pISSN - 1615-9853
DOI - 10.1002/pmic.200601029
Subject(s) - polystyrene , polymer , chemistry , chromatography , peptide , methyl methacrylate , surface modification , methacrylate , polymer chemistry , copolymer , organic chemistry , biochemistry
A novel protocol of rapid and automatic on‐plate desalting (OPD) and peptide concentration for 2‐DE‐MALDI‐MS has been developed by the approach of templating the hydrophobic polymer solution over Kapton‐etched mask. For the template technique, small hydrophobic polymer [linear poly(methyl methacrylate) (PMMA), PMMA derivatized with fullerene‐C 60 (PMMA‐C 60 ), linear polystyrene (PSt), or PSt derivatized with fullerene‐C 60 (PSt‐C 60 )] spots (990 μm od) are patterned at the centers of stainless MALDI plate wells (1400 μm id). Tryptic–peptide solution with no predesalting was dropped onto the central hydrophobic spots, resulting in a concentration of proteolytic peptides on the hydrophobic polymer surface with a reduced spot size. The dried peptide layer was then covered subsequently with over‐volume matrix solution, causing the removal of redissolved salts from the spot center to the spot edge by means of a natural “outward flow.” The proposed OPD protocol exhibited a dramatic enhancement in S/N up to 850 for 14 fmol BSA digests in the coexistence of 100 mM salts, compared with barely detectable peaks in ordinary way. This analysis has shown that the success rate of identification was increased by two‐fold for low abundance proteins in the human liver tissue with no need for the conventional ZipPlate desalting strategy.