Premium
Fractionation and separation of human salivary proteins by pH‐gradient ion exchange and reversed phase chromatography coupled to mass spectrometry
Author(s) -
Pepaj Milaim,
Holm Anders,
Fleckenstein Burkhard,
Lundanes Elsa,
Greibrokk Tyge
Publication year - 2006
Publication title -
journal of separation science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.72
H-Index - 102
eISSN - 1615-9314
pISSN - 1615-9306
DOI - 10.1002/jssc.200500346
Subject(s) - chromatography , chemistry , elution , fractionation , isoelectric point , mass spectrometry , two dimensional chromatography , isoelectric focusing , ion exchange , monolithic hplc column , analytical chemistry (journal) , high performance liquid chromatography , ion , biochemistry , organic chemistry , enzyme
Abstract In the present work, a 2‐D capillary liquid chromatography method for fractionation and separation of human salivary proteins is demonstrated. Fractionation of proteins according to their p I values was performed in the 1‐D employing a strong anion exchange (SAX) column subjected to a wide‐range descending pH gradient. Polystyrene‐divinylbenzene (PS‐DVB) RP columns were used for focusing and subsequent separation of the proteins in the 2‐D. The SAX column was presaturated with a high pH buffer (A) consisting of 10 mM amine buffering species, pH 9.0, and elution was performed with a low pH elution buffer (B) having the same buffer composition and concentration as buffer A, but pH 3.5. Isoelectric point fractions eluting from the 1‐D column were trapped on PS‐DVB trap columns prior to back‐flushed elution onto the PS‐DVB analytical column for separation of the proteins. The 1‐D fraction eluting at pH 9.0–8.7 was chosen for further analysis. After separation on the RP analytical column, nine RP protein fractions were collected and tryptic digested for subsequent analyses by MALDI TOF MS and column switching capillary LC coupled to ESI TOF MS and ESI QTOF MS. Eight proteins and two peptides were identified in the pH 9.0–8.7 fraction using peptide mass fingerprinting and uninterpreted MS/MS data.