Mass spectrometry, posttranslational modifications analysis

Top-down proteomics [1] is the identification and characterization of a mature, intact protein (or proteins) using primarily mass spectrometry (MS)-based techniques and the sequence information contained in genomic/proteomic databases. Unhke bottom-up proteomics [2-4], where a protein (or proteins) is digested into peptides prior to MS or tandem mass spectrometry (MS/MS) analysis [3,4], the top-down approach involves measuring the molecular weight (MW) of the intact, mature protein with its associated posttranslational modifications (PTMs) if any. The intact protein ion is then fragmented in the gas phase in order to determine its amino acid sequence as well as the location and identification of PTMs. From its earliest development, top-down proteomics has been primarily the domain of electrospray ionization (ESI) [5] (which generates mul-... 

The results for bacterial whole-cell analysis described here establish the utility of MALDI-FTMS for mass spectral analysis of whole-cell bacteria and (potentially) more complex single-celled organisms. The use of MALDI-measured accurate mass values combined with mass defect plots is rapid, accurate, and simpler in sample preparation then conventional liquid chromatographic methods for bacterial lipid analysis. Intact cell MALDI-FTMS bacterial lipid characterization complements the use of proteomics profiling by mass spectrometry because it relies on accurate mass measurements of chemical species that are not subject to posttranslational modification or proteolytic degradation. 

Larsen, M.R., Trelle, M.B., Thingholm, T.E., and lensen, O.N. 2006. Analysis of posttranslational modifications of proteins by tandem mass spectrometry. BioTechniques 40, 790-798. 

Mass spectrometry is more than 100 times more accurate than gel electrophoresis in molecular mass determination, and obtaining the data for sequence analysis requires only a fraction of the time needed for Edman degradation. Moreover, mass spectrometry is well suited for analysis of posttranslational modifications, which cannot be determined by Edman degradation. 

The determination of the molecular mass of the peptides generated after proteolysis is called bottom-up or peptide-level spectroscopy. In contrast, the measurements of intact proteins are called top-down or intact protein-level spectrometry. These two methods are not mutually exclusive but are complementary and essential to provide the complete picture of the amino acid sequence and many posttranslational modifications of a protein. These two terms, i.e., bottom-up and top-down, are borrowed from genomics and are analogous to such terms used in the DNA sequencing during genome analysis. 

Because of the advances in the gas-phase ionization of biomacromolecules, such as electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI), mass spectrometry (MS) has become a powerful tool for detection, identification, and structural analysis of proteins, peptides, and polynucleotides. The molecules ionized in a gas phase by these methods are subsequently analyzed by sector, quadrupole, ion-trap, or time-of-flight mass spectrometers. In particular, the MS systems consisting of ESI and triple-stage quadrupole (ESI/TSQ) or ion-trap (IT) mass spectrometry and MALDI time-of-flight (MALDl/TOF) mass spectrometry have been most widely applied to the field of protein chemistry for the accurate determination of molecular mass of proteins and peptides, determination of amino acid sequence, identification of proteins by peptide mass databases, and analysis of posttranslational modifications such as phosphorylation and glycosylation. In general, current techniques allow detenni-... 

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