Proteomic profiling of a snake venom using high mass detection MALDI-TOF mass spectrometry

Abstract

Proteomic profiling involves identification and quantification of protein components in complex biological systems. Most of the mass profiling studies performed with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) have been restricted to peptides and small proteins (Ͻ20 kDa) because the sensitivity of the standard ion detectors decreases with increasing ion mass. Here we perform a protein profiling study of the snake venom Sistrurus miliarius barbouri, comparing 2D gel electrophoresis and reversed-phase high-performance liquid chromatography (HPLC) with a high mass cryodetector MALDI-TOF instrument (Macromizer), whose detector displays an uniform sensitivity with mass. Our results show that such MS approach can render superior analysis of protein complexity compared with that obtained with the electrophoretic and chromatographic approaches. The summation of ion impacts allows relative quantification of different proteins, and the number of ion counts correlates with the peak areas in the reversed-phase HPLC. Furthermore, the sensitivity reached with the high mass cryodetection MS technology clearly exceeds the detection limit of standard high-sensitivity staining methods. (J Am Soc Mass Spectrom 2007, 18, 600 -606) © 2007 American Society for Mass Spectrometry P rotein profiling has become a powerful method for analyzing changes in global protein expression patterns in biological systems as a function of developmental, physiological, and disease processes. Two-dimensional (2D) gel electrophoresis has been established for many years as the primary tool for detecting proteins present in an organism or a complex biological extract. However, 2D gel electrophoresis has some limitations: limited solubility of hydrophobic and membrane proteins, limited dynamic range, difficulty in focusing highly basic and acidic proteins, poor sensitivity, poor quantitation, and finally, the method is not amenable to automation. The limitations associated with gel electrophoretic analysis of peptides and small proteins (Ͻ20 kD) have stimulated interest in mass spectrometry as an alternative strategy. In this sense, MALDI-TOF mass spectrometry has partially replaced or complemented gel-based approaches for studying the proteomic profile of the low mass components of many complex biological samples such as serum [1] or tissues [2]. Imaging MS [2] and chip-based technologies such as surfaceenhanced laser desorption/ionization (SELDI-MS) [3] are relatively new approaches that take advantage of the established features of MALDI MS (mass accuracy, sensitivity, reliability, and high-throughput) to detect and identify peptides and proteins. Also, 1D and 2D liquid chromatography coupled to detection systems such as UV and/or MALDI, have been gradually introduced for the analysis and profiling of intact (top down proteomics) [4,5] and digested proteins (bottom up proteomics) [6] in complex biological mixtures. MALDI is a soft ionization method and produces predominantly singly charged molecular ions r...

Reproducibility in Protein Profiling by MALDI-TOF Mass Spectrometry

“…The matrix molecules produce highly intense signals in the low-mass range (ϳ0 -1000 (6 ). The 3rd challenge in MALDI protein profiling is establishing the reproducibility of peak intensity.…”

MALDI imaging and profiling MS of higher mass proteins from tissue

“…In contrast, the mass spectrum obtained with the high mass HM1 detector contains many peaks up to m/z 50,000 ( Figure 1b). Several studies have established that the detection sensitivity of higher mass proteins using an MCP detector is highly attenuated in multi-component mixtures, due to detector saturation [14,17,24,25]. The tissues analyzed in a MALDI imaging and profiling MS analysis contain a very large number of proteins that will exacerbate this loss of sensitivity due to detector saturation.…”

“…A particular advantage of STJ detectors is that multiply charged ions can be distinguished on the basis of their higher kinetic energy. The higher detection efficiency and higher capacity of the STJ detector has been exploited for the analysis of complex protein mixtures: it was demonstrated that the STJ detector could detect multiple higher mass proteins that were not detected using an MCP [24]. Unfortunately the Comet Macromizer is no longer commercially available.…”

The concept of two-dimensional electrophoresis-guided purification proven by isolation of rhodocetin from Calloselasma rhodostoma (Malayan pit viper)

“…Since its first description thirty years ago, twodimensional gel electrophoresis (2DE) has been the primary tool for detecting proteins in an organism and complex biological extracts (1,2). By separating proteins based on two independent parameters -pI values by isoelectric focusing (IEF) in one dimension and molecular mass by SDS-PAGE in the other -samples can be resolved to a few thousand protein spots (3).…”