Mass spectrometry and proteomics.

THE 15TH ASILOMAR CONFERENCE on Mass Spectrometry this October was devoted to the role of mass spectrometry (MS) in proteomics. The Asilomar Conference site is in a picturesque national park in Pacific Grove, CA, overlooking the Pacific Ocean. The conference aims to bring together scientists from a cross section of disciplines that are applying MS to an emerging field. This year, that emerging field is proteomics. The term ‘‘proteome’’ was coined by Wilkins et al. (17) in the mid-1990s to describe the protein complement of the genome. The term was first used to describe the 20-yrold field of two-dimensional gel electrophoresis (2-DE) and quantitative image analysis. 2-DE remains the highest resolution protein separation method available, but the ability to identify the observed proteins was always an extremely difficult problem. MS has been integral to solving that problem.Although improvements in 2-D gel technology had been realized since its introduction, three enabling technological advances have provided the basis for the foundation of the field of proteomics. The first advance was the introduction of large-scale nucleotide sequencing of both expressed sequence tags (ESTs) and, more recently, genomic DNA. The second was the development of mass spectrometers able to ionize and mass-analyze biological molecules and, more recently, the wide-spread introduction of mass spectrometers capable of data-dependent ion selection for fragmentation (MS/MS)(i.e., without the need for user intervention). The third was the development of computer algorithms able to match uninterpreted (or partially interpreted) MS/MS spectra with translations of the nucleotide sequence databases, thereby tying the first two technological advances together. Thus MS played a key role in the passage of 2-DE/image analysis to proteomics. As a note to readers unfamiliar with MS, the instruments are named for their type of ionization source and mass analyzer (see also Refs. 1, 11, 12). To measure the mass of molecules, the test material must be charged (hence ionized) and desolvated (dry). The two most successful mechanisms for ionization of peptides and proteins are matrix-assisted laser desorption ionization (MALDI) and electrospray ionization (ESI). In MALDI the analyte of interest is embedded in a matrix that is dried and then volatilized in a vacuum under ultraviolet laser irradiation. This is a relatively efficient process that ablates only a small portion of the analyte with each laser shot. Typically, the mass analyzer coupled with MALDI is a time-of-flight (TOF) mass analyzer that simply measures the elapsed time from acceleration of the charged (ionized) molecules through a field-free drift region. The other common ionization source is ESI, in which the analyte is sprayed from a fine needle at high voltage toward the inlet of the mass spectrometer (which is under vacuum) at a lower voltage. The spray is typically either from a reversedphase HPLC (RP-HPLC) column or a nanospray device (19) that is similar to a microinjection needle. During this process, the droplets containing analyte are dried and gain charge (ionize). The ions formed during this process are directed into the mass analyzer, which could be either a triple-quadrupole, an ion trap, a Fourier-transform ion cyclotron resonance (FT-ICR), or a hybrid quadrupole TOF (Qq-TOF) type. This Asilomar meeting provided one of the largest academic forums in the United States for the presentation and discussion of MS as it is applied to proteomics. As is obvious from the introduction, the initial role MS played was as a protein identification and characterization methodology. However, the role of MS is expanding in this field. Although a series of talks focused on the use of different kinds of MS to identify gel-separated proteins and the various automation technologies applied to perform this in high throughput, several talks also presented alternate approaches. These approaches utilized direct analysis of digested protein mixtures for either identification of the components or quantitative analysis of two different samples mixed together. Specific biological applications were also presented. As described above, a critical component of any MS approach as applied to proteomics is the computational analysis. This report will be divided to focus on these six aspects of MS in proteomics. Where references are known for some of the material presented, they are cited. The program was, however, not entirely limited to MS in proteomics. Prior to the six sections covering the conference core, the first section of this report covers those presentations that were aimed at providing an insight into broader biological and drug discovery processes. Proteomics in biology and drug discovery. The opening lecture, given by Lee Hood (Univ. of Washington), provided an excellent overview of Genomics, Proteomics, and Systems Biology. Hood described the genome project efforts that provide four types of maps: genetic, physical, gene, and sequence. For the human genome, Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org).