P1: FUMMay 9, 2001 14:6 Annual Reviews AR131-14Annu. Rev. Biochem. 2001. 70:437–73Copyrightc° 2001 by Annual Reviews. All rights reservedANALYSIS OF PROTEINS AND PROTEOMES BYMASS SPECTROMETRYMatthias Mann,1,2Ronald C. Hendrickson,2and Akhilesh Pandey31Protein Interaction Laboratory and Center for Experimental BioInformatics (CEBI),Department of Biochemistry and Molecular Biology, University of Southern Denmark,Campusvej 55, 5230 Odense M, Denmark; e-mail: [email protected] Proteomics A/S, Staermosegaardsvej 6, 5230 Odense M, Denmark;e-mail: [email protected] Institute for Biomedical Research, 9 Cambridge Center, Cambridge,Massachusetts 02142, and Brigham and Women’s Hospital, Boston, Massachusetts 02115;e-mail: [email protected] Words functional genomics, signal transduction, posttranslationalmodifications, protein interaction, phosphorylation, proteomics■ Abstract A decade after the discovery of electrospray and matrix-assisted laserdesorption ionization (MALDI), methods that finally allowedgentleionization of largebiomolecules, mass spectrometry has become a powerful tool in protein analysis andthe key technology in the emerging field of proteomics. The success of mass spectrom-etry is driven both by innovative instrumentation designs, especially those operatingon the time-of-flight or ion-trapping principles, and by large-scale biochemical strate-gies, which use mass spectrometry to detect the isolated proteins. Any human proteincan now be identified directly from genome databases on the basis of minimal dataderived by mass spectrometry. As has already happened in genomics, increased au-tomation of sample handling, analysis, and the interpretation of results will generate anavalanche of qualitative and quantitative proteomic data. Protein-protein interactionscan be analyzed directly by precipitation of a tagged bait followed by mass spectro-metric identification of its binding partners. By these and similar strategies, entireprotein complexes, signaling pathways, and whole organelles are being characterized.Posttranslational modifications remain difficult to analyze but are starting to yield togeneric strategies.CONTENTSINTRODUCTION ................................................ 438IONIZATION METHODS.......................................... 440MALDI...................................................... 440Electrospray................................................... 4400066-4154/01/0701-0437$14.00437P1: FUMMay 9, 2001 14:6 Annual Reviews AR131-14438 MANN¥HENDRICKSON¥PANDEYMASS SPECTROMETERS ......................................... 442MALDI Time-of-Flight Mass Spectrometer............................ 442Electrospray Quadrupole Mass Spectrometers.......................... 442Ion-Trapping Instruments......................................... 444Other Mass Spectrometers........................................ 445PROTEIN PURIFICATION AND PREPARATION........................ 445Special Considerations for Protein Preparation Methods................... 445Digestion and Preparation of Gel-Separated Proteins for MS Analysis......... 446ANALYSIS OF INTACT PROTEINS.................................. 446PEPTIDE SEQUENCING BY TANDEM MASS SPECTROMETRY........... 448PROTEIN IDENTIFICATION BY DATABASE SEARCHING................ 450Peptide Mass Fingerprinting....................................... 450Searching with Tandem Mass Spectrometric Data........................ 450Searching Protein, Expressed Sequence Tag, and Genome Databases.......... 453Database Searching and Protein Modifications.......................... 454LIQUID CHROMATOGRAPHY AND TANDEM MASS SPECTROMETRY..... 454Principles.................................................... 454Instrumental and Practical Considerations............................. 455Recent Advances in Instrumentation and Strategy........................ 458ANALYSIS OF POSTTRANSLATIONAL MODIFICATIONS................ 459QUANTITATIVE MASS SPECTROMETRY............................. 462PROTEOMIC STRATEGIES AND EXAMPLES.......................... 463Differential 2-D Gels Followed by Mass Spectrometric Protein Identification.... 463Protein Interactions and Protein Complexes............................ 464Signal Transduction Pathways...................................... 466Secreted Proteins............................................... 467Analyzing Protein Complexes and Organelles Without Gel Separation......... 468OUTLOOK..................................................... 470INTRODUCTIONMass spectrometry (MS) is a venerable technique whose beginnings date back tothe early days of the last century. Among the analytical techniques, MS holds aspecial place because it measures an intrinsic property of a molecule, its mass,with very high sensitivity and therefore it is used in an amazingly wide rangeof applications. Beginning in the 1980s and on a larger scale in the 1990s, massspectrometry has played an increasingly significant role in the biological sciences.Why has it taken so long? Mainly because mass spectrometers require charged,gaseous molecules for analysis. Biomolecules being large and polar, however,they are not easily transferred into the gas phase and ionized. Electrospray (ES)(1) and matrix-assisted laser desorption ionization (MALDI) (2) are the ionizationtechniques that should be credited most for the success of mass spectrometry inthe life sciences. These methods were developed in the late 1980s and were thebasis for the increasingly powerful instrumentation that became available a fewP1: FUMMay 9, 2001 14:6 Annual Reviews AR131-14PROTEINS ANALYSIS BY MASS SPECTROMETRY 439years later. Major advanceswere also made in sample preparation for MS, a crucialarea for overall feasibility and sensitivity of analysis. Starting in 1993, softwarealgorithms were published that allowed the correlation of mass spectrometric dataobtained for a protein with the increasingly populated sequence databases. Inretrospect, this event marked the transformation of mass spectrometry into a large-scale, functional genomics technique. The last few years have seen development ofeven more powerful instrumentation and algorithms for protein characterization,a trend that shows no signs of slowing down.At the same time MS was being developed to meet the demands of molec-ular biology for high sensitivity, the concept of proteomics began to be pop-ularized. Proteomics is now defined as the large-scale analysis of the functionof genes and is becoming a