Regardless of the choice of a given proteomic separation technique, gel-based or gel-free, a mass spectrometer is always the primary tool for protein identification. During the last decade, significant improvements have been made in the application of MS for the determination of protein sequences [161] . Mass spectrometers consist of an ion source, the mass analyzer, and an ion detection system. Analysis of proteins by MS occurs in three major steps (a) protein ionization and generation of gas-phase ions, (b) separation of ions according to their mass to charge ratio, and (c) detection of ions [162] . In gel-free approaches such as ICAT and MudPIT, samples are directly analyzed by MS whereas, in gel-based proteomics (2DE and 2D-DIGE), the protein spots are first excised from the gel and then digested with trypsin. The resulting peptides are then separated by LC or directly analyzed by MS. The experimentally derived peptide masses are correlated with the peptide fingerprints of known proteins in the databases using search engines (e.g., Mascot, Sequest). There are two main ionization sources which include matrix assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) and four major mass analyzers, which are time-of-flight (TOF), ion trap, quadrupole, and fourier transform ion cyclotron (FTIC) which are currently in use for protein identification and characterization [163] . A combination of different mass analyzers in tandem such as quadrupole-TOF and quadrupole-ion trap has combined the individual strengths of different types of mass analyzers and greatly improved their capabilities for proteome analysis [162] . Simple mass spectrometers such as MALDI-TOF are used for only measurement of mass, whereas tandem mass spectrometers are used for amino acid sequence determination [164] . In MALDI the sample of interest is crystallized with the matrix on a metal surface and a laser ion source causes excitation of matrix along with the analyte ions, which are then released into the gas phase. MALDI measures the mass of peptides derived from a trypsinized parent protein and generates a list of experimental peptide masses, often referred to as "mass fingerprints" [165, 166] . In ESI, the analyte is ionized from a solution and transferred into the gas phase by generating a fine spray from a high voltage needle which results in multiple charging of the analyte and generation of multiple consecutive ions. Tandem mass spectrometry or MS/MS is performed by combining two different MS separation principles. In tandem MS, individual trypsin-digested peptides are fragmented after a liquid phase separation. Tandem MS instruments such as triple quadrupole, quadrupole ion trap, fourier transform ion-cyclotron resonance, or quadrupole time-of-flight are used in LC-MS/MS or nanospray experiments with electrospray ionization (ESI) to generate peptide fragment ion spectra [167] . Ion mobility spectrometry (IMS) has been utilized as a rapid gas-phase separations strategy for biomolecular ions [168, 169] . The strategy provides high sensitivity because the gas-phase dispersion of peptide ions separates features corresponding to low abundance species from interfering chemical noise [170] . Reduced spectral congestion also allows for the use of shorter experimental run times (LC separations) without sacrificing throughput; short analysis time scales are key to measuring the large numbers of samples required to determine normal protein variability prior to realizing individual plasma profiling. Additionally, mobility-dispersed ions can be fragmented and mobility linked to fragment ions without ion loss from precursor mass selection [171] . These advantages have been demonstrated in head-to-head comparisons with conventional LC-MS/MS technology using rapid (21 minutes) LC gradients [169] . Accurate mass and time (AMT) tag approach [172] addresses an analogous situation in LC-MS-based proteomics studies. In this approach, initial LC-MS/MS analyses are performed on prefractionated peptide samples in order to provide peptide sequence identifications. These experiments are relatively low throughput because the peptide prefractionation can be quite extensive and require separate LC-MS/MS analyses for each fraction. The high-throughput accurate mass and time (AMT) tag proteomic approach was utilized to characterize the proteomes for cytoplasm, cytoplasmic membrane, periplasm, and outer membrane fractions from aerobic and photosynthetic cultures of the gram-negative bacterium Rhodobacter sphaeroides 2.4.1. There has been a recent trend in proteomics toward the development and application of technologies for the targeted analysis of proteins within complex mixtures [173] . Selected reaction monitoring (SRM) is a powerful tandem mass spectrometry method that can be used to monitor target peptides within a complex protein digest [174, 175] . The specificity and sensitivity of the approach, as well as its capability to multiplex the measurement of many analytes in parallel, has made it a technology of particular promise for hypothesis driven proteomics. The use of tandem mass spectrometry data acquired on an LTQ ion trap mass spectrometer can accurately predict which fragment ions will produce the greatest signal in an SRM assay using a triple quadrupole mass spectrometer [176] . One of the biggest benefits of a targeted assay on a triple quadrupole mass spectrometer is high throughput. Using the selectivity of multiple stages of mass selection of a tandem mass spectrometer, these targeted SRM assays are the mass spectrometry equivalent of a Western blot [173] . An advantage of using targeted mass spectrometry-based assay over a traditional Western blot is that it does not rely on the creation of any immunoaffinity reagent. While its application is novel in the proteomics community, SRM has been utilized for several decades in the toxicology and pharmacokinetics disciplines [177] . Peptidebased immunofractionation methods show potential for proteome wide screening approaches but are limited by the availability of antibodies [178, 179] . The stable isotope standards with capture by antipeptide antibodies (SISCAPA) approach is based on the addition of stable isotope labeled standard peptides to the digested clinical sample followed by immunoaffinity enrichment of standard and analyte peptide by highly specific antipeptide antibodies [180, 181] . This approach enables the absolute quantification of selected diagnostic peptides from digested clinical samples down to physiologically relevant analyte concentrations (ng/mL) at high precision (10% CV) and accuracy [178, 179] . Further improvement of MRM-based biomarker quantification should be possible if whole sets of analyte peptides can be enriched by immunofractionation. Since this method relies on one specific antibody per target protein/peptide the generation of more than 10000 antibodies is necessary for proteome wide screening approaches. Novel peptide affinity enrichment strategies enabling proteome wide analyses of signature peptides may provide an important addition to future proteome workflows. Undoubtedly, the accuracy, high throughput, and robustness of MS technologies have made the characterization of entire proteomes a realistic goal [180, 181] .