Analysis of proteins and glycoproteins at the picomole level by on-line coupling of microbore high-performance liquid chromatography with flow fast atom bombardment and electrospray mass spectrometry: A comparative evaluation

Abstract

A comparison of continuous-flow fast atom bombardment (FAB) mass spectrometry and nebulization-assisted electrospray for analysis of proteins and glycoproteins by high-performance liquid chromatography (HPLC)/mass spectrometry is presented. The evaluation was made using enzymatic digests of recombinant soluble CD4 glycoprotein and recombinant hepatitis B surface antigen and a mixture of HPLC retention standard peptides. These samples were analyzed by reversed-phase HPLC on a microbore (1 x 100 mm C18) gradient HPLC system with 10% or 20% of the eluent containing approximately 20-100 pmol of the sample directed into the continuous-flow FAB mass spectrometric or electrospray mass spectrometric source, respectively. The techniques were evaluated on the criteria of chromatographic integrity, ease of data analysis, protein sequence coverage, discrimination effects, ability to detect glycopeptides, simplicity of operation, and sensitivity. Both techniques produce useful peptide molecular weight data from comparable amounts of sample injected. However, the nebulization-assisted electrospray system is capable of yielding higher peptide mapping coverages with the least sample consumed in toto due in part to the wider mass ranges resulting from the multicharging effect and to the ability to detect glycopeptides. Under the experimental conditions employed here no fragmentation was observed in the electrospray mass spectra. In contrast, significant, sequence informative fragmentation was occasionally observed for peptides in the continuous-flow FAB mass spectral data.