Proteomics and Related Applications in Oral Cancer and Sjögren's Syndrome

Mass Spectrometry-Based Proteomics

Modern proteomics is primarily driven by mass spectrometry (MS), an exquisite analytical technology which measures the mass-to-charge ratio of ionized molecules. A commonly used approach for proteomic studies is based on 2-D gel electrophoresis (2-DE) with MS. In this approach, proteins are initially mapped out by 2-DE or 2-D differential gel electrophoresis (DIGE). To identify a proteins of interest, the protein spot is excised for in-gel digestion and the resulting peptides are analyzed using matrix-assisted laser desorption/ionization (MALDI) or electrospray (ESI) with time-of-flight MS (TOF MS) or more sophisticated tandem MS. The obtained masses are then in silico compared to either a proteome or genome database to find the best matched protein. This is achieved by using a computer program for database searching (e.g., MASCOT or SEQUEST), which calculates the absolute masses of theoretical peptides from each protein in the database and then compares the peptide masses of the unknown protein to the theoretical peptide masses of each protein in the database. The protein identification method based on MALDI-TOF MS or ESI-TOF MS with database searching is also known as peptide mass fingerprinting (PMF) (Pappin, 1993; Mann, 1993, Yates, 1993). Typically PMF is for the identification of an isolated protein. The presence of a protein mixture can significantly complicate the analysis and potentially compromise the results. Therefore, a mixture of 2 or more proteins typically requires the use of tandem MS to achieve confident identification of the proteins.

Tandem MS, also known as MS/MS, involves multiple stages of MS analysis, with some form of fragmentation occurring in between the stages (Aebersold & Mann, 2003). Tandem MS can be done in space by using physically separated mass analyzers with a collision cell between these elements for molecule fragmentation. For example, one mass analyzer can isolate a peptide ion from many entering a mass spectrometer. The peptide is then broken into smaller fragments in the collision cell by collision-induced dissociation (CID) and a second mass analyzer measures the fragment ions produced from the peptide. Tandem MS can also be done in time. Ions are trapped in a single mass analyzer with multiple MS steps taking place over time, as in a quadruple ion trap or Fourier transform MS (FT-MS) instrument.

Shotgun proteomics refers to the direct analysis of complex protein mixtures to rapidly generate a global profile of the protein complement. It mainly relies on tandem MS and database-searching algorithms for peptide and protein identification. The shotgun approach has been facilitated by the use of multidimensional protein identification technology (MudPIT), which incorporates 2-D liquid chromatography (2-D LC), ESI-MS/MS, with SEQUEST database searching (Washburn, 2001; Wolters, 2001). Due to its high resolving power and compatibility with ESI-MS, 2-D LC represents a primary separation method for peptides in proteomics. To realize an in-depth proteome analysis, other liquid separation techniques such as capillary electrophoresis, zoom isoelectric focusing, free-flow electrophoresis, can be used for prefractionation of proteins prior to 2-D LC-MS/MS to achieve a more comprehensive proteomic analysis (Guo, 2006; Zuo, 2006; Xie, 2006).