Mass spectrometry for proteomics

Abstract

Mass spectrometry has been widely used to analyze biological samples and has evolved into an indispensable tool for proteomics research. Our desire to understand the proteome has led to new technologies that push the boundary of mass spectrometry capabilities, which in return has allowed mass spectrometry to address an ever-increasing array of biological questions. The recent development of a novel mass spectrometer (Orbitrap) and new dissociation methods such as electron-transfer dissociation has made possible the exciting new areas of proteomic application. Although bottom-up proteomics (analysis of proteolytic peptide mixtures) remains the workhorse for proteomic analysis, middle-down and top-down strategies (analysis of longer peptides and intact proteins, respectively) should allow more complete characterization of protein isoforms and post-translational modifications. Finally, stable isotope labeling strategies have transformed mass spectrometry from merely descriptive to a tool for measuring dynamic changes in protein expression, interaction, and modification.

Figure 1

Strategies for MS-based protein identification and characterization. Proteins extracted from biological samples can be analyzed by bottom-up or top-down methods. In the bottom-up approach, proteins in complex mixtures can be separated prior to enzymatic (or chemical) digestion followed by direct peptide mass fingerprinting-based acquisition or further peptide separation on-line coupled to tandem mass spectrometry. Alternatively, the protein mixture can be directly digested into a collection of peptides (“shotgun” approach), which are then separated by multidimensional chromatography on-line coupled to tandem mass spectrometric analysis. In the top-down approach, proteins in complex mixtures are fractionated and separated into pure single proteins or less complex protein mixtures, followed by off-line static infusion of sample into the mass spectrometer for intact protein mass measurement and intact protein fragmentation. An on-line LC-MS strategy can also be used for large scale protein interrogation.