Most questions in modern cell biology have been approached using reductionist methods, i.e., by studying one gene, one protein or one specific protein modification at a time. This reductionism has been necessary, given the complexity of biological systems and lack of tools for developing more integrative methodologies. It is, nonetheless, responsible for most of our knowledge of biological systems. However, a more thorough understanding of complex systems will require the simultaneous observation of their many characteristics. Proteomic methodologies, especially those that are mass spectrometry based, have enabled the large-scale study of protein modifications, protein abundance and protein interactors. Only by using such integrative approaches will we develop rigorous models that more accurately reflect actual cellular processes.

Proteomics is the study of the set or subset of all proteins expressed in an organism, tissue or cell culture. While proteomics is by no means a novel idea, recent advances in mass spectrometry and the determination of the complete genomic sequences of several organisms greatly enhance its usefulness [1–3]. Only by mass spectrometry can one efficiently identify the individual protein components derived from protein complexes, and only large-scale genomic data allows amino acid sequences to be reliably assigned to peptide fragments identified by mass spectrometry.

Most proteomics-based studies today seek to answer four basic questions: Which proteins were found? What is the relative abundance of the proteins found? What modifications were found on the proteins? Which proteins physically associate with one another? One type of proteomics experiment, protein profiling, involves the identification of the proteins present in a complex, cell culture, tissue or organism. However, unlike genomes, proteomes are dynamic with changes that reflect their current functional state. Thus, analyzing changes in the abundance and modifications of proteins in a complex could be considered a functional study (functional proteomics). While several proteomic-based methods exist, most are based on a standard sequence of experiments: a protein complex is obtained by biochemical prefraction-ation or affinity purification; the sample is subjected to enzymatic digestion (usually trypsin); the resulting peptides can be labeled for future analysis; and the peptides are analyzed by mass spectrometry to determine their identity and characteristics.

At the core of most modern proteomic studies lies mass spectrometry (MS) [2,3]. A mass spectrometer is, in essence, a detector that measures the mass-to-charge ratio (m/z) of ionized particles and detects the relative number of ions at each m/z ratio. In general, peptide fragments generated by enzymatic cleavage (typically trypsin) are ionized most commonly by matrix-assisted laser desorption/ionization (MALDI) or electrospray ionization (ESI) and injected into a mass spectrometer. In most cases, peptides with specific m/z ratios can be selected by quadrupole exclusion or time of flight (TOF) selection. These peptides can then be fragmented by cleavage at peptide bonds via collision with a gaseous matrix. The masses of the fragments can then be determined by a second mass analysis (often by TOF or ion trap MS) to determine the amino acid sequence of peptides. Prefractionation of samples by nanoflow high-performance liquid chromatography (HPLC) and increases in sensitivity and accuracy of mass spectrometers allows for the identification of up to 1000 or more peptides per sample. In this way, complex mixtures can be resolved and the protein compositions elucidated.