Comparison of the capabilities of liquid isoelectric focusing–one-dimensional nonporous silica reversed-phase liquid chromatography–electrospray ionization time-of-flight mass spectrometry and liquid isoelectric focusing–one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis mass mapping for the analysis of intact protein molecular masses

Abstract

Nonporous silica reversed-phase HPLC coupled to electrospray ionization with on-line time-of-flight mass spectrometric detection (NPS-RP-HPLC–ESI-TOF-MS) is shown to be an effective liquid phase method for obtaining the molecular masses of proteins from pH fractionated cellular lysates where the method is capable of generating the same banding patterns typically observed using gel phase one-dimensional sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The liquid-phase mass spectrometry-based method provides a mass accuracy of at least 150 ppm, with 4000 mass resolution and provides improved sensitivity as the protein molecular mass (MW) decreases. The liquid and gel phase methods are shown to be complementary in terms of their mass range but the liquid phase method has the advantage over the gel method in that the analysis times are 50 times shorter, the mass accuracy is 70 times better and the resolution is 130 times higher. The liquid phase method is shown to be more effective for detection of proteins below 40 kDa, while the gel phase separation can access many more proteins, including more hydrophobic proteins, at increasing MW.