Isoform separation of a multi-acetylated protein using capillary polystyrene-divinylbenzene monolithic columns.
Capillary polystyrene-divinylbenzene (PS-DVB) monolithic columns were used to separate differentially acetylated intact IM9 protein isoforms. Compared to the unmodified form, the hydrophobic shift for intact acetylated isoforms was significant under standard reversed-phase conditions (32.5-45% acetonitrile in 10 min). The high chromatographic resolution of the PS-DVB monolithic columns resulted in peak widths at half height of 4-5s. This allowed us to nearly completely resolve a number of peaks greater than the number of possible acetylation sites. This observation suggested that not only the number, but also the location of the acetylations on the protein had a significant effect on the retention. Matrix-assisted laser desorption ionization time-of-flight MS and MS/MS were used to confirm the chromatographic separation of isoforms. It was found that the acetylations site, especially on the N-terminus, has an effect on the retention on the PS-DVB column.
Sneekes
Evert-Jan
EJ
Dionex Corporation, Amsterdam, The Netherlands. Evert-Jan.Sneekes@dionex.com
Damen
Mirjam
M
Swart
Remco
R
Heck
Albert J R
AJ
D000107:Acetylation; D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation* / Q000379:methods*; D011137:Polystyrenes / Q000737:chemistry*; D020033:Protein Isoforms / Q000737:chemistry / Q000302:isolation & purification; D011506:Proteins / Q000737:chemistry / Q000302:isolation & purification*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D053719:Tandem Mass Spectrometry
D011137:Polystyrenes; D020033:Protein Isoforms; D011506:Proteins; C432006:divinylbenzene-polystyrene copolymer