Matrix-assisted laser desorption/ionization-mass spectrometry of hydrophobic proteins in mixtures using formic acid, perfluorooctanoic acid, and sorbitol.
Three MALDI-MS sample/matrix preparation approaches were evaluated for their ability to enhance hydrophobic protein detection from complex mixtures: (1) formic acid-based formulations, (2) perfluorooctanoic acid (PFOA) surfactant addition, and (3) sorbitol addition. While MALDI-MS of Escherichia coli cells desorbed from a standard sinapinic acid matrix displayed 94 (M + H)+ ions, 119 were observed from a formic acid-based matrix with no more than 10 common to both. Formic acid matrix revealed many lipoproteins and an 8282 m/z ion proposed to be the abundant, water-insoluble ATPase proteolipid. Among the formic acid-based cocktails examined, the slowest rate of serine/threonine formylation was found for 50% H2O/33% 2-propanol/17% formic acid. Faster formylation was observed from cocktails containing more formic acid and from mixtures including CH3CN. Sinapinic, ferulic, DHB, 4-hydroxybenzylidene malononitrile, and 2-mercaptobenzothiazole matrixes performed well in formic acid formulations. Dramatic differences in mixture spectra were also observed from PFOA/sinapinic acid, at detergent concentrations exceeding the critical micelle concentration, although these matrix cocktails proved difficult to crystallize. E. coli ions observed from these matrix conditions are listed in Tables S-1 and S-3 (Supporting Information). Similar complementarity was observed for M. acetivorans whole-cell mixtures. Including sorbitol in the sinapinic acid matrix was found to promote homogeneous crystallization and to enhance medium and higher m/z ion detection from dilute E. coli cellular mixtures.
Loo
Rachel R Ogorzalek
RR
Department of Biological Chemistry and Department of Chemistry & Biochemistry, Molecular Biology Institute, UCLA-DOE Institute for Genomics & Proteomics, University of California-Los Angeles, Los Angeles, California 90095, USA. rloo@mednet.ucla.edu
Loo
Joseph A
JA
D000251:Adenosine Triphosphatases / Q000032:analysis; D001426:Bacterial Proteins / Q000032:analysis*; D002210:Caprylates; D002477:Cells / Q000737:chemistry; D003460:Crystallization; D029968:Escherichia coli Proteins / Q000032:analysis; D005466:Fluorocarbons; D005561:Formates; D057927:Hydrophobic and Hydrophilic Interactions; D008074:Lipoproteins; D017020:Methanosarcina / Q000737:chemistry; D013012:Sorbitol; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D001426:Bacterial Proteins; D002210:Caprylates; D029968:Escherichia coli Proteins; D005466:Fluorocarbons; D005561:Formates; D008074:Lipoproteins; C030544:formic acid; D013012:Sorbitol; C023036:perfluorooctanoic acid; D000251:Adenosine Triphosphatases