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.