Applications of mass spectrometry to signal transduction

PERGAMON

Progress in Biophysics & Molecular Biology 71 (1999) 501±523

Applications of mass spectrometry to signal transduction Katheryn A. Resing a, Natalie G. Ahn a, b, * a

Department of Chemistry and Biochemistry, University of Colorado, Boulder, CO 80309, USA b Howard Hughes Medical Institute, University of Colorado, Boulder, CO 80309, USA

Abstract Advances in mass spectrometry instrumentation, protocols for sample handling, and computational methods provide powerful new approaches to solving problems in analytical biochemistry. This review summarizes recent work illustrating ways in which mass spectrometry has been used to address questions relevant to signal transduction. Rather than encompass all of the instruments or methodologies that might be brought to bear on these problems, we present an overview of commonly used techniques, promising new methodologies, and some applications. # 1999 Elsevier Science Ltd. All rights reserved.

1. Instrumentation One of the major reasons for success of mass spectrometry in analyzing biomolecules is the introduction of soft ionization techniques to `volatilize' proteins, peptides, oligonucleotides (reviewed by Chapman, 1996). After charging, ions are drawn into the mass spectrometer by an electric ®eld gradient, where their mass to charge ratio is measured. In electrospray ionization (ESI), charged droplets are produced by passing a solubilized sample through a high voltage needle at atmospheric pressure (Fenn et al., 1989). Desolvation of the charged droplets prior to entrance into the high vacuum region of the mass spectrometer typically occurs as a consequence of passage through a warm dry countercurrent of gas or through a heated capillary. Desorption of analyte ions occurs in the electric ®eld as the droplets desolvate. ESI typically induces a range of charge forms, ([M + 1]/1, [M + 2]/2, [M + 3]/3, etc.) consistent with the chemical nature of the analyte. In matrix assisted laser desorption ionization (MALDI), samples are cocrystallized onto a sample plate with a small organic matrix compound that absorbs at the wavelength of the laser (Hillenkamp et al., 1991). This is * Corresponding author. Department of Chemistry and Biochemistry, Campus Box 215, University of Colorado, Boulder, CO 80309, U.S.A. Tel.: +1-303-492-4799; fax: +1-303-492-2439; e-mail: [email protected] 0079-6107/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved. PII: S 0 0 7 9 - 6 1 0 7 ( 9 8 ) 0 0 0 4 8 - 0

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followed by laser excitation of the matrix to desorb both matrix and analyte, inducing ionization by an unknown mechanism. MALDI produces primarily singly charged ions, although multiply charged ions are sometimes seen, particularly with protein ions. ESI and MALDI ionization procedures are combined in various ways with di€erent mass analyzers, which detect either negative or positively charged ions. Four types are most often used with biomolecules: Quadrupole mass analyzers resolve mass/charge by varying radio and electromagnetic frequencies within the analyzer, so as to allow only a narrow window of mass/ charge to reach the detector. Commercially available instruments have detection ranges from zero to 04000 m/z and are normally set at unit resolution1, with higher resolution possible at the expense of sensitivity. In ESI, multiply charging of ions enable molecules up to 100,000 Da to be detected within the observable window. Time-of-¯ight (TOF) analyzers accelerate the ions in a short voltage gradient and measure the time they take to traverse a ®eld free ¯ight tube; the ¯ight time is proportional to the square root of the mass/charge. Recent advances in delayed extraction (Edmondson and Russell, 1996) and re¯ectron technologies (Niehuis et al., 1987), that focus ions by correcting for variations in kinetic energy and spatial distributions, have increased resolution in the small mass range >10,000. Because the focusing ability of TOF instruments is inversely proportional to ¯ight time, resolution on larger biomolecules is lower. Quadrupole ion trap mass spectrometers focus ions into a small volume with an oscillating electric ®eld; ions are resonantly activated and ejected by electronic manipulation of this ®eld (reviewed in Jonscher and Yates, 1997). Inexpensive quadrupole ion traps, relying primarily on RF ®elds, have recently been introduced. Fourier transform ion cyclotron resonance (FTICR) mass spectrometers use high magnetic ®elds to trap the ions, and cyclotron resonance to detect and excite the ions (Amster, 1996), with resolution of 100,000. The sensitivity of ion trap instruments is inherently higher because samples at low concentration can be allowed to accumulate within the quadrupole ion traps over a few microseconds, or inde®nitely for an FTICR. With FTICR, detection of 10±50 amol (10 ÿ 18 mol) of proteins and peptides has been reported (Solouki et al., 1995; Valaskovic et al., 1996). One of the ®rst con®gurations widely useful for biological applications was ESI interfaced with a quadrupole mass analyzer, which allows sample application to be directly coupled to reversed-phase HPLC (LC/MS) (Covey et al., 1988). Collision induced dissociation (CID) to produce diagnostic fragmentation can be induced at the ori®ce; with tandem quadrupoles, ions can be selected in one quadrupole and activated by collisionally induced dissociation (CID) in a second quadrupole, producing fragment ions which are then analyzed in a third quadrupole (MS/MS or MS2). Together, the method enables sequencing of peptides or oligonucleotides within complex mixtures such as proteolytic or nuclease digests and provides a means of identifying modi®ed residues or bases. Other biomolecules such as lipids and carbohydrates can be identi®ed by their characteristic fragmentation patterns. Fragmentation and sequencing of dicult or large ions is further enhanced using quadrupole ion trap instruments, which trap fragment ions for further cleavage, thus enabling successive CID experiments (MSn) to be performed on a single sample. Sequencing of proteins up to 28 kDa have been reported using 1

Mass resolution is de®ned as M/DM, where M is mass and DM is the width of the observed ion peak at half maximum peak height (FWHM).

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an FTICR; although this is time consuming and computationally dicult, no theoretical limits exist on size (Smith et al., 1996). The most common combination of MALDI is with a TOF mass spectrometer, providing a wide window of observable masses (as high as 106 m/z), femtomole sensitivity, and simpler spectra containing only singly and some doubly charged ions. Delayed extraction and re¯ectron technologies allow fragment ions to be observed, thus enabling MALDI-TOF instruments to accommodate sequencing by fragmentation using either post-source decay (PSD) which relies on metastable decomposition of ions enroute to the detector (Spengler, 1996), or in-source decay (Lennon and Walsh, 1997). Some instruments have collision cells, allowing CID as well. ESI is also available combined with TOF, in order to take advantage of the high scan speed and the resolution in the low mass range. Hybrid instruments combining di€erent mass analyzers are becoming available that take advantage of the strengths of each. For example, the quadrupole TOF is expected to enhance MS/MS analysis by combining the high ion selectivity of a quadrupole with high resolution analysis of the fragments in the TOF (Morris et al., 1996). Because mass spectrometers are concentration-dependent analyzers (the concentration of the protein/peptide determines signal intensity, not the amount of sample applied), methods to minimize sample volume and the ¯ow rate of sample application facilitate sensitivity of detection. Adoption of capillary HPLC (i.d. 0.075±0.5 mm) has increased sensitivity to the 100 femtomole range or even better (Huang and Henion, 1991) and driven development of low ¯ow techniques for HPLC. Recently, low ¯ow nanospray or micro-ionspray techniques have been introduced (Wilm and Mann, 1996), which combined with methods for sample concentration, enable detection at 10±100 fmol/ml in ideal cases. Sequence analyses of biological samples, combining nanospray techniques with quadrupole or quadrupole ion trap mass analyzers, have now been reported by several labs (Wilm et al., 1996; McCormack et al., 1997). Capillary electrophoresis coupled to instruments with fast scan speed (TOF or ion trap) is another promising approach for high sensitivity analysis of complex mixtures (Severs and Smith, 1997), although many applications require specially constructed capillary columns that currently are not available commercially.

2. Analysis of protein posttranslational modi®cations Mass spectrometry is ideally suited for mapping protein post-translational modi®cations (PTMs). A major advantage of mass spectrometry is that the analysis of PTMs is unbiased, because they are observed by alterations in protein or peptide mass. Thus is it possible to detect unusual or unexpected chemistries. A list of known modi®cations is available at the website developed by Ken Mitchelhill (http://www.abrf.org/ABRF/Research Committees/ deltamass/deltamass.html). 2.1. Methods MS was ®rst applied to identi®cation of phosphorylation sites using fast atom bombardment ionization and a four sector mass analyzer (Cohen et al., 1991). These protocols required

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separate puri®cation of phosphopeptides prior to analysis, usually by RP-HPLC, and were limited in mass range and sensitivity (100 pmol at best). However, they provided a useful alternative to Edman degradation and paved the way for applications to later instruments. In the simplest approach, ecient mapping of PTM sites is attained by proteolytic digestion of proteins and mass analysis of digest components. Modi®ed peptides are identi®ed by comparing observed masses to those predicted from expected peptide sequences, and when possible, by comparing peptide masses between digests of modi®ed vs. unmodi®ed proteins. This is followed by PSD or CID peptide sequencing to deduce the modi®ed residues based on predicted changes in fragment ion masses (Annan and Carr, 1996; Resing and Ahn, 1997; Kuster and Mann, 1998). Phosphorylation analysis has had the greatest impact on signaling studies, and will be used as an example of the problems of experimental design that arise during mass spectrometric analysis of PTMs. In complex digests, identifying PTMs often proves dicult, particularly if the stoichiometry of modi®cation is low (as often observed in response to signaling, where components of a pathway may only be partially activated). Gas phase chemical reactions can be exploited to identify the modi®ed peptides. For example, two ESI/MS scanning methods exist for selectively detecting phosphopeptides. One utilizes the propensity for neutral loss of phosphoric acid after collisional activation, scanning for decreases in m/z of all ions between two mass analyzers of a tandem instrument (Covey et al., 1991). An alternative method uses high ori®ce voltage conditions to generate fragment ions, PO3ÿ (79 Da/e) and PO2ÿ (63 Da/e), which are identi®ed by scanning in negative ion mode (Huddleston et al., 1993; Carr et al., 1996). Both scanning methods have been successfully adapted to nanospray infusion, enabling detection of low abundance phosphopeptides. Alternatively, modi®ed peptides can be selectively puri®ed. In the case of phosphopeptides, immobilized metal anity chromatography (IMAC) using Fe3+loaded chelating resins can be used (Watts et al., 1994; Yip and Hutchens, 1996). Studies of multiple phosphopeptides in the cystic ®brosis transmembrane conductance regulator using both ion scanning and IMAC techniques showed that the results from these selection methods were roughly comparable, but both were less sensitive than direct positive ion mode LC/MS (Neville et al., 1997; Townsend et al., 1996). An understanding of the chemistry of the PTM is essential to development of appropriate analytical protocols. Collisional activation for sequencing or PSD often leads to neutral loss of phosphoric acid (ÿ98 Da) from phosphoserine or phosphothreonine from phosphoserine, rather than cleavage at peptide bonds. Chemical beta-elimination of phosphate to convert phosphoserine and phosphothreonine respectively to dehydroalanine and dehydrothreonine recovers the eciency of peptide fragmentation. Alternatively, the peptide can be sequenced, even after neutral loss has occurred, by sequential ion selection and fragmentation using an ion trap MS, bypassing the need for chemical treatment. Analysis of phosphohistidine is complicated by its acid lability and increased susceptibility to hydrolysis during analysis. Protocols for optimizing detection and sequencing of phosphohistidine containing peptides use neutral bu€ers during LC and nonacidic matrices for MALDI (Medzihradszky et al., 1997). High sequence coverage of modi®able residues over the entire protein is needed to ensure that all sites have been identi®ed. Usually, greater than 90% coverage can be attained with judicious selection of one or more proteases. Quadrupole ion trap or FTICR instruments can fragment larger peptides than triple quadrupole collision cells, enabling analysis of larger

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polypeptides, for example, intact proteins or those produced by cleavage at rare amino acids such as Trp or Met, which should facilitate analysis of PTMs. 2.2. Applications There are now a few hundred examples in which mass spectrometry has been used to locate PTMs within signaling proteins by analyzing proteolytic digests. Examples include phosphorylation, N- and O-glycosylation (Burlingame, 1996; Greis et al., 1996; Neumann et al., 1998), regulated proteolytic processing (Resing et al., 1993), oxidation (Dow et al., 1997), disul®de crosslinking (Sun et al., 1996), acetylation (Lin et al., 1998), ADP ribosylation (Zhou et al., 1996), lipid modi®cation (Pepinsky et al., 1998), and S-nitrosylation (Matthews et al., 1996). In a noteworthy example, an inactivation mechanism for the small GTPase Rho, was identi®ed from a mass change of 1 Da due to conversion of Gln to Glu (Schmidt et al., 1997). Particularly exciting are advances in structure determination of complex glycoconjugates by MALDI-TOF (Harvey, 1996). Mass spectrometry should eventually reveal details of changes in extracellular carbohydrate structure observed in response to cell signaling and di€erentiation (Hanisch et al., 1996), an area that has been previously neglected due to the heterogeneity and poor solubility of glycocalyx components. In cases where protein sequences are known, the protein masses can be measured and PTMs detected by deviations from predicted masses. Examples include analyses of ribosomal proteins (Louie et al., 1996) and photosynthetic reaction center proteins (Sharma et al., 1997). A widespead use of this application is to document structural heterogeneity of puri®ed proteins due to variability in PTMs (e.g., glycosylation of rhodopsin, Whitelegge et al., 1998) or sequence (e.g. ®laggrin, Resing et al., 1993). Cellular proteins can also be screened for speci®c PTMs by metabolic radiolabelling of intact cells with labelled precursors, visualizing proteins by SDS-PAGE/autoradiography, and sequencing excised proteins (as discussed below). This method has been used to identify protein phosphorylation in thrombin-stimulated platelets (Immler et al., 1998), prenylation in lens tissue (Cenedella, 1998), and covalent adducts of acetaminophen with liver proteins in drug treated rats (Qiu et al., 1998). Multiple phosphorylation sites are often encountered on signaling molecules, where the phosphorylation events can be regulated in a complex manner, and better methods are needed to analyze the regulatory relationship between these protein modi®cations. Quantitative measurements of the kinetics of modi®cation is feasible using ESI-MS, because the ionization eciencies of modi®ed vs. unmodi®ed peptides are usually similar enough to estimate stoichiometries. Three studies illustrate the use of mass spectrometry to dissect complex behavior of multiple phosphorylation events; analogous approaches can be applied to other PTMs. In the ®rst example, kinetics of autophosphorylation of c-src at Tyr338, Tyr419, and Tyr530 were measured in vitro (Boerner et al., 1996). By quantifying peptides containing each site, the individual kinetics of phosphorylation could be monitored simultaneously by LC/MS and compared with speci®c activities measured in parallel. The results show enzyme activation and inactivation upon phosphorylation of Tyr419 and Tyr530 respectively. In the second example, LC/MS and MS/MS analysis of protein kinase CbII phosphorylation combined with di€erential sensitivity to phosphatases were used to delineate the hierarchy of phosphorylation

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events that regulate activation and cellular relocalization of enzyme (Keranen et al., 1995). Thr500 phosphorylation by an unknown kinase enables autophosphorylation at Thr641, which in turn facilitates autophosphorylation at Ser660, and modi®cation of Ser660 alters subcellular localization. In the third example, hierarchal phosphorylation of three sites in rhodopsin, located within one tryptic peptide, were monitored by MS (Ohguro et al., 1993). ESI-MS revealed mono-, diand tri-phosphorylated forms of the peptide, with phosphate at Thr334, Ser338, and Ser343. Evidence for hierarchal phosphorylation was indicated by the appearance of only one monophosphorylated peptide, modi®ed at Ser338, and two diphosphorylated peptides, modi®ed either at Ser338/Thr336 or Ser338/Ser343. In the future, this simple analysis may be extended to more complicated phosphorylation patterns, by utilizing MSn capabilities of ion trap or FTICR spectrometers to sequence larger peptides or even intact proteins. For example, the microtubule binding protein, stathmin/Op18 exists in at least twelve di€erentially phosphorylated forms, only three of which have been characterized (Zugaro et al., 1998). The ability to determine the order and stoichiometry of modi®cation at widely separated sites of modi®cation will undoubtedly reveal new insights about regulatory mechanisms in signaling.

3. Proteomics and protein complexes High instrument sensitivity combined with recent low ¯ow and low volume sample handling protocols, now allow analysis of proteins at levels detectable in silver stained gels. Ecient ingel proteolysis of proteins resolved by 1D or 2D SDS-PAGE and extraction of peptides for MS analysis shows great promise for characterization of signaling pathways. For example, these strategies facilitate direct sequencing and identi®cation of components in protein complexes of low abundance, such as signaling or transcription complexes. This provides a major advantage over immunoprecipitation and immunoblotting, and other strategies that depend on the availability of probes and educated guesses about protein constituents. Peptide mass analysis and MS sequencing from in-gel digests have also brought new excitement to a ®eld referred to as `proteomics', which in analogy to genomics, determines the total expression pattern of cellular proteins (Shevchenko et al., 1996; Humphery-Smith and Blackstock, 1997; Celis et al., 1998; Yates, 1998). 2D electrophoretic maps of extracts from cells treated under di€erent conditions reveal changes in proteins that are complementary to methods of di€erential display, subtractive hybridization, or expressed sequence tag identi®cation. This is especially useful for signal transduction studies, where the visualization of 2D gels provides a means of rapidly inspecting total cellular proteins for changes in expression, and additionally o€ers information about changes in posttranslational modi®cations in response to cell activation. 3.1. Methods In-gel digests are performed by excising a gel piece containing a protein of interest, decolorizing and dehydrating the gel, then rehydrating with a bu€er containing protease. Most protocols use acetylated trypsin, which is resistent to autolysis and allows longer digestions to

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be carried out, minimizing contamination by peptides derived from trypsin. Peptides are extracted by passive elution, and either analyzed by capillary LC/MS or desalted and concentrated for analysis by MALDI, nanospray, or micro-ionspray MS. Negative staining using Zn2+-imidazole is as sensitive as silver staining and generally gives higher yield of peptides (Castellanos-Serra et al., 1996). Elution of the protein from the gel before digestion is also possible, but increases sample handling. Alternatively, proteins can be electroblotted to membranes for digestion, although yields are often lower (Courchesne et al., 1997). For protein identi®cation, information about the masses of peptides in digests and/or partial sequencing of several peptides is obtained in order to search sequence databases (Shevchenko et al., 1997b). Several programs are available for these analysis; especially useful are the web based search engines at EMBL (http://www.mann.embl-heidelberg.de/services/peptidesearch/ peptidesearchintro.html) and USCF (http://prospector.ucsf.edu). Some search programs make use of `tag sequences', short amino acid sequences derived from MS/MS data, which, along with the masses of fragments containing the N- and C-terminal unsequenced portion of the peptide, may provide sucient information to identify a protein Ð in ideal cases, from only 2± 3 residues. Sequence tags from two or more peptides will usually yield an unequivocal identi®cation of the protein. When using peptide masses alone to search databases (`peptide mass ®ngerprinting'), the accuracy of mass determination is a limiting factor. Typically, several candidate proteins are obtained in such searches, accounting for a small subset of the input peptide masses. After masses of peptides identi®ed in the ®rst search are used to recalibrate the mass spectrum, other peptides can usually be revealed, increasing the likelihood of protein identi®cation. The driving force behind these strategies is the increasing probability that a given unknown can be identi®ed from translated cDNA sequence or expressed sequence tag databases, which for human proteins is estimated at 80%. In cases where the protein is absent from databases, sequencing can provide sucient information for cloning. The sequencing of peptides from unknown proteins (de novo sequencing) requires tag sequences of 6±7 amino acids that correspond to low redundancy oligonucleotides. A critical problem with this approach is the common appearance in tag sequences of Ile or Leu, which have the same mass and cannot be distinguished easily. Other complications include reduced cleavage near Gly, leading to ambiguities between Gly±Gly and Asn, and between Gly±Ala and Gln. Cysteines can also become alkylated with acrylamide, therefore it is advisable to reduce and alkylate samples before SDS-PAGE. Proteome analyses began in the 1980's with the development of high resolution protocols for 2D electrophoretic separation. As proteins were identi®ed by Edman microsequencing, a few laboratories began assembling databases of observed pI and molecular weights of proteins from human tissues (Celis et al., 1995, 1998). The application to signal transduction was immediately apparent, and studies were reported describing changes in protein expression or pI between extracts of cells treated with various growth factors and mitogens (Levenson and Blackshear, 1989; Levenson et al., 1989; Schwartz, 1993). Sequencing methods at that time lacked the sensitivity to identify low abundance proteins and did not have the high throughput necessary for exhaustive proteome analysis. However, MS approaches are now at detection levels which allow analysis of proteins in abundance of 100±1000 copies/cell (10±100 fmol). The development and commercial availability of immobilized pH gradient (IPG) strips has

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been particularly important for IEF separations with high reproducibility and resolving power (Corbett et al., 1994; Righetti and Bossi, 1997). Protein mobility can be localized with respect to other protein markers using various computer programs for 2D mapping, such as Melanie II (Appel et al., 1997) or PDQUEST (Rasmussen et al., 1996), allowing comparison with the proteome databases. Many proteome databases are publically available at web sites; a particularly useful one is the SWISS-2DPAGE database accessible at the ExPASy molecular biology server in Geneva (http:/expasy.hcuge.ch/www/expasy-top.html). Non-gel based IEF methods under development, such as capillary IEF (Figeys and Aebersold, 1998) may eventually replace 2D electrophoresis with faster and less labor intensive protocols. 3.2. Applications 3.2.1. Subunits within protein complexes A widely used and very successful application of 1D or 2D electrophoretic separation and mass spectrometric sequencing is in the identi®cation of subunits within stable protein complexes puri®ed by chromatographic fractionation or di€erential centrifugation. Many studies are performed in yeast, where open reading frame sequences of the corresponding genes can be rapidly identi®ed. Examples in single cell eukaryotes include new protein components in Saccharomyces cerevisiae spindle pole complexes (Wigge et al., 1998), U1 snRNP (Neubauer et al., 1997) and the anaphase promoting complex/ubiquitin-protein ligase (Zachariae et al., 1998), as well as the identi®cation of histone acetyltransferases bound to TAFs in stable transcription complexes (Grant et al., 1998), and the catalytic subunit of Euplotes telomerase ribonucleoprotein complex (Lingner et al., 1997). Examples in multicellular eukaryotes include identi®cation of protein components in the IkB kinase (IKK) signaling complex (Mercurio et al., 1997), Drosophila chromatin-accessibility complex (Varga-Weisz et al., 1997), human nuclear matrix (Holzmann et al., 1997), and MHC class I chaperone complexes (Lindquist et al., 1998). In signaling systems, complexes involving receptors and downstream signaling components can be less stable and dicult to purify. Anity or immunoanity techniques are used in such cases to identify cellular proteins that associate in vitro with DNA or protein targets (Yates et al., 1997). For example, coimmunoprecipitation of 35 S-labelled proteins with the CD95/Fas/ APO-1 receptor and MS sequencing led to the identi®cation of the FLICE protease as a component of the death-inducing signaling complex (Muzio et al., 1996). Identi®cation and sequencing of extract proteins that coimmunoprecipitate with Gza have revealed Gg and Gb subunits as well as phospholipase C-g (Bartlett and Hendry, 1997). Protein-coupled anity resins have also been used to identify a novel protein, Rabex-5, as one of several proteins that associate with Rab5 (Horiuchi et al., 1997), a T cell receptor interacting molecule (TRIM) that binds to the TCR in response to signal activation (Bruyns et al., 1998), IQGAP1 as a predominant calmodulin binding protein in human breast cancer cells (Joyal et al., 1997), binding of a Dictyostelium STAT transcription factor to an oligonucleotide promoter response element (Kawata et al., 1997), interaction of prohibitin with the SH3 domain of mixed lineage kinase 2 (Rasmussen et al., 1998), and binding of cellular proteins to SHC (Patterson et al., 1996).

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3.2.2. Proteome applications Proteomic strategies are easiest to perform in organisms whose genomic sequences are known, thus providing a complete database for sequence comparison and peptide ®ngerprinting. Several databases exist for prokaryotes, for example, Escherichia coli (VanBogelen et al., 1997), Hemophilus in¯uenzae (Langen et al., 1997; Link et al., 1997) and Spiroplasma melliferum (Cordwell et al., 1997). Correlations between protein expression and cell type- or strain-dependent pathogenicity or changes in varying environmental conditions has practical applications for identifying unique markers as potential new drug targets. In one study, identi®cation of proteins not predicted from genome information suggested errors in the interpretation of nucleic acid sequencing results (Link et al., 1997). A combination of gene knockout and proteome analysis has also proven useful to identifying the function of translated genes from genome sequencing studies. Examples are adaptation of E. coli to sulfate starvation (Dainese et al., 1997) and response of Bacillus subtilis to temperature and chemical stresses (Antelmann et al., 1997). Saccharomyces cerevisiae also provides a good model system for proteome mapping (Shevchenko et al., 1996; Larsson et al., 1997) and for examining eukaryotic cell responses to signaling. In one example, changes in protein expression in response to osmotic stress were examined following treatment with high salt, which has the cellular e€ect of elevating glycerol synthesis (Blomberg, 1997). Of the twenty most strongly induced proteins were nine enzymes involved in glycerol metabolism, including one of previously unknown function that was subsequently characterized biochemically as a glycerol 3-phosphatase involved in glycerol biosynthesis. This illustrates the utility of proteomic strategies for identifying the function of translated genes from genomic sequences. Proteomics in mammalian cells lacks the advantage of completed genome sequences; nevertheless, much work is already aimed at comparing normal and diseased states of tissues and cell lines, with the goal of identifying speci®c tissue markers that can be used in diagnoses or as drug targets. Mammalian proteome databases reported so far include those for human keratinocytes (Celis et al., 1995), myocardial tissue and hypertrophic cardiac myocytes (Sutton et al., 1995; Thiede et al., 1996), bladder carcinomas (Rasmussen et al., 1996; éstergaard et al., 1997), colorectal cancer cell lines (Ji et al., 1997), mouse liver cells (O'Connell and Stults, 1997), and rat serum proteins (Haynes et al., 1998). Other mammalian databases are organelle speci®c, examining proteins from nuclei (Nilsson et al., 1997), Golgi vesicles (Shevchenko et al., 1997a) and mitochondria (Rabilloud et al., 1998), isolated from human cells. Only a handful of studies have utilized proteomic approaches combined with mass spectrometry to examine changes in proteins in response to speci®c signaling events or pathways. Examples include identi®cation of proteins induced in antiproliferative responses of human renal and cervical carcinoma and melanoma cells to interleukin 4, interferon-g, and tumor necrosis factor (Epstein et al., 1996; Matsui et al., 1997; Sullivan et al., 1997), and stress inducible proteins in human cells (Vogt et al., 1996). So far, most of the proteins that have been identi®ed in mammalian cell proteome studies are abundant (e.g. cytoskeletal proteins, metabolic enzymes, heat shock proteins) and are more likely to be important as markers of downstream responses, rather than as causal regulators of disease or di€erentiation. However, future work in this area, including further analyses of protein changes, improvements in methods for separation and MS detection, and completion

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of genomic databases, may eventually yield mechanistic insight into cellular responses. It will be particularly interesting to observe how closely the changes in kinetics and total levels of protein expression will compare to the behavior of their corresponding mRNA transcripts. Ultimately, strategies of this type can be applied to cells in which speci®c signaling pathways are stimulated or blocked through expression of signaling molecules in active or inactive forms. Such information can be used to establish the global behavior of downstream responses to speci®c signaling pathways, from which we can begin to address questions that are currently dicult to answer, such as how cells of the same type respond di€erently to di€erent pathways, how di€erent cell types respond di€erently to the same pathway, and how di€erent signaling pathways interact synergistically to elicit new cellular responses.

4. Lipid signaling An important application of mass spectrometry is characterizing and quantifying molecules of low molecular weight in intact cells and organisms. This has great potential for studying the regulation of signaling events, for example, by monitoring levels of lipid second messengers and determining their structures. Furthermore, many of the proteomic changes observed following cell transformation involve metabolic enzymes, and it is clear that cell di€erentiation can profoundly in¯uence speci®c metabolic pathways. For example, 1a,25dihydroxycholecalciferol is primarily metabolized by the C24 oxidation pathway in undi€erentiated human colon adenocarcinoma-derived cell lines, whereas in di€erentiated cells, it is metabolized by a di€erent route to 1a,25-dihydroxy-3a-cholecalciferol (Bischof et al., 1998). Methods of analyzing small molecule compounds by MS should facilitate measurements of metabolic intermediates as indicators of downstream responses to signaling pathways. 4.1. Methods A typical analytical protocol for lipids involves HPLC puri®cation, derivatization, and mass analysis by gas chromatography/mass spectrometry (GC/MS). CID is commonly carried out on fast atom bombardment mass spectrometers to generate diagnostic fragmentation patterns. FAB/MS is still commonly used, particularly when large amounts of material are available. However, FAB often does not ionize highly acidic compounds well, and only produces singly charged ions, so that larger conjugated lipids are outside the instrument m/z range. In recent years, studies have reported that LC/MS provides superior sensitivity and ease of use, as the sample usually need not be derivatized, and also provides increased analyte stability. Methods for analysis of lipids and glycolipids by ESI/MS have recently been reviewed (Ohashi, 1997). An advantage of triple quadrupole instruments is that product ion scanning can be used to identify speci®c lipids within complex mixtures, by scanning for diagnostic CID-generated fragment ions (`multiple reaction monitoring'). For example, thermolabile lipoxygenase-derived fatty acid hydroperoxides were analyzed by scanning for neutral loss of hydrogen peroxide (Schneider et al., 1997). This study also demonstrates the use of adduct ion formation as an alternative to derivatization for analysis of uncharged components, where in the presence of ammonium acetate, hydroperoxide lipids form ammoniated ions [M + NH4] + . In another

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study, structural characterization of phosphatidylcholine was achieved by CID of [M + Cl] ÿ ions in negative-ion electrospray mass spectra (Lehmann et al., 1997). Even structurally simple, neutral molecules such as polypropylene glycol will often form charged ammonia or metal ion adducts. The neutral retinoids, retinol, and retinal, can be observed because these conjugated molecules bind protons even through there are no formal chargeable groups (Van Breemen and Huang, 1996). As with protein applications, it is anticipated that new methods for concentrating samples will arise to exploit the sensitivity of ESI and MALDI mass spectrometers. For example, solid-phase extraction on membrane disks has been used to quantify leukotriene E4 in human urine (Wu et al., 1996). LC/MS and CID fragmentation have been used in identi®cation of ceramides from human leukemia HL-60 cells (Couch et al., 1997), sphingomyelin and dihydrosphingomyelin from human lens membranes (Byrdwell and Borchman, 1997), and mitogenic phospholipids generated after corneal injury (Liliom et al., 1998). A study directly comparing the ionization of aminophospholipids by ESI vs. liquid secondary ion/electric-magnetic sector ionization demonstrated that ESI was remarkably superior in sensitivity, but that the high-energy collision magnetic sector ionization yielded better fragmentation for structural information (Chen, 1997). It seems likely that the MSn capabilities of ion traps will provide fragmentation comparable to that of the sector instruments. Furthermore, the use of LC/MS enables analysis of lipid modi®cations on proteins in complex systems. Analysis of the corni®ed envelope of epidermal cells, where cross-linking of ceramides to membrane proteins plays an important role in barrier function in both normal and disease states, simultaneously identi®ed ceramide esteri®cation and the sites of modi®cation on involucrin, periplakin and envoplakin (Marekov and Steinert, 1998). Ideally, one would like to look at individual cells in intact tissue. In this light, recent work on secondary ion mass spectrometry (SIMS) shows exciting promise for studies of small molecule metabolism and signaling in tissues (Todd et al., 1997). In SIMS, an ion beam rasters across a tissue section, ionizing small molecules or generating fragment ions (secondary ions) directly from the surface, which are then detected in a mass spectrometer. In one study, a triple quadrupole mass detector was used to characterize distribution of phosphatidylcholine in brain and spleen tissue sections (Todd et al., 1997); development of an ion trap or time-of-¯ight SIMS instrument would likely improve the sensitivity of these studies. An exciting new technique is MALDI ion image analysis, in which tissue sections directly analyzed or blotted onto adsorbant material are coated with a thin layer of matrix and scanned, providing 2dimensional imaging and localization of biomolecules in various regions of tissue (Caprioli et al., 1997). 4.2. Applications A signi®cant amount of work has analyzed lipid signaling and metabolism during the in¯ammatory response. Central to the understanding of in¯ammation is synthesis of arachidonic acid and its metabolism to prostaglandins and hydroxyeicosatetraenoic acids by cyclooxygenase, lipoxygenase and cytochrome P450 monooxygenase-dependent enzymes. Analysis of PGE2, 12-HETE, and arachidonic acid has been carried out in extracts of cultured human dermal ®broblasts (Newby and Mallet, 1997), as well as epoxyeicosatrienoic and

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monohydroxyeicosatetraenoic acids esteri®ed to phospholipids in human red blood cells (Nakamura et al., 1997). The scope of analysis can be quite broad: in one study, 14 eicosanoids were quanti®ed in single samples from in¯ammatory models of carrageenan-challenged rat air pouch and lipopolysaccharide-stimulated human whole blood, demonstrating high amounts of prostaglandin E2, TxB2, and 6-keto prostaglandin F1a, and lower amounts of prostaglandins E1, D2, and F2a and leukotrienes B4 and C4 (Margalit et al., 1996). LC/MS enabled identi®cation of novel metabolites of prostaglandin E2 formed by isolated rat hepatocytes, where a striking observation was that previous studies had overlooked taurine conjugates that comprised nearly 50% of the mixture of metabolites (Hankin et al., 1997). Several examples have appeared relating glycolipid or lipid metabolism to problems in signaling. Ceramide and sphingolipid metabolites have been implicated as second messengers in studies of mammalian cell apoptosis. LC/MS detection of sphingomyelin demonstrates a role for the sphingomyelin-ceramide cycle during Fas-induced apoptosis of rat ovarian follicle cells (Foghi et al., 1998). MS combined with functional cell studies led to the characterization of gangliosides shed by neuroblastoma tumors which downregulated cellular immune responses, thereby enhancing the ability of tumors to avoid immune surveillance (Li et al., 1996). ESI/MS has also been used to show accumulation of gangliosides in testis and epididymis due to targeted disruption of Hex B beta-hexosaminidase in mouse models of Tay Sachs and Sandho€ diseases (Trasler et al., 1998). In yeast, sphingolipids act as second messengers to signal accumulation of the thermoprotectant, trehalose. MS analysis of lipids in sphingolipid-de®cient strains of Saccharomyces cerevisiae that are unable to resist heat shock suggests that heat resistance involves de novo ceramide synthesis (Wells et al., 1998). LC/MS has been used to show that cyclosporin A reduces biliary phosphatidylcholine excretion, presumably via inhibition of phosphatidylcholine translocation across the hepatocyte canalicular membrane by the Mdr2 P-glycoprotein (Lehmann et al., 1997). MS analysis of cell culture metabolites has shown that following deprivation of L-serine, sphingolipids and phosphatidyl-L-serine are reduced while phosphatidyl-L-threonine is synthesized by hippocampal neurons, supporting a role of L-serine in survival and neuritogenesis of hippocampal neurons (Mitoma et al., 1998).

5. Protein structure Various features of protein structure can be examined using mass spectrometric protocols, including protein±protein or protein±ligand interactions as well as protein conformation and folding. Many of these applications rely on classical strategies in protein chemistry, in which the mass accuracy and sequencing capability of MS provides a signi®cant advantage over SDSPAGE and Edman degradation. In addition, it is often not appreciated that methods are available to examine hydrogen exchange behavior in proteins, providing information on folding kinetics and protein dynamics in solution. 5.1. Protein±protein and protein±ligand interactions Subunit interfaces within noncovalent protein complexes are mapped by comparing e€ects of limited proteolysis on proteins in bound vs. unbound states. LC/MS provides a rapid method

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to screen for protected regions by di€erential cleavage, and peptide products are unequivocally identi®ed by LC/MS/MS. Results can be veri®ed by covalent crosslinking between subunits or by inhibiting complex formation with peptide competition. Examples in which subunit interactions have been mapped within protein complexes relevant to signaling are the syntaxin 1A/SNAP-25/vesicle-associated membrane protein 2 (VAMP2) complex involved in synaptic vesicle exocytosis (Poirier et al., 1998), and the vinculin/E-cadherin/a-catenin adherens junction complex (Weiss et al., 1998). Alternative approaches have been put forth, including a technique often applied to antibody epitope mapping, in which one protein is digested and the peptide products are incubated with immobilized antibody. Unbound peptides are removed and bound peptides are eluted and analyzed by MS. In ideal cases, a single peptide is identi®ed, and synthetic peptides with sequence variations are tested to de®ne residues critical for recognition (Zhao et al., 1996). Anity extraction has also been used to identify peptide ligands that interact with speci®c class I or class II MHC receptors (Hayden et al., 1996; Tomlinson et al., 1996). This strategy can also be applied to positive or subtractive screening of peptide libraries (Lyubarskaya et al., 1997; Yu et al., 1997). Another method useful for conformationally dependent epitopes is to de®ne the protected epitope surface area by chemically modifying residues on the surface of the protein before proteolysis, screening for di€erences in modi®cation as changes in peptide mass upon comparing free antigen to the antigen±antibody complex (Fiedler et al., 1998). Promising new applications under development use mass spectrometry to screen for candidate inhibitors from combinatorial libraries that can be enriched by stable noncovalent binding with protein targets (Nedved et al., 1996; Kaur et al., 1997; Van Breemen et al., 1997). Screening methods for phosphorylated products from peptide libraries have also been used to de®ne substrate speci®cities for protein kinases (Till et al., 1994) and protein phosphatases (Huyer et al., 1998), and to determine recognition sequences for SH2 domains (Kelly et al., 1996). Mass spectrometry can also be applied to mapping ligand binding sites on receptors and other proteins. Because of the relatively low anity for most such binding interactions, these methods usually require crosslinking between ligand and receptor, followed by proteolysis and identi®cation of crosslinked residues by MS. Crosslinked peptides can be enriched before MS by modifying peptide ligands with ¯uorescent tags and monitoring ¯uorescence by RP-HPLC, or by coupling ligands to biotin and puri®cation on streptavidin-linked resins. Examples where ligand binding sites have been mapped by MS include chemoattractant N-formyl peptide receptor (Mills et al., 1998), neurokinin-1 receptor (Girault et al., 1996), and G-CSF receptor (Haniu et al., 1995). 5.2. Conformation, folding, and dynamics Classic strategies of partial proteolysis or chemical modi®cation combined with MS analysis of products have been used to probe changes in solution conformation of signaling proteins. For example, proteolytic susceptibility has been used to probe conformational e€ects of OmpR phosphorylation (Kenney et al., 1995), and changes in glycinamidation of carboxyl groups has been used to reveal di€erences in the tertiary structure between GDP and GTP bound forms of Ras in the presence of Raf-1 (Akashi et al., 1997). These techniques can also be used to probe

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folding by comparing native to denatured molecular states, as in proteolytic mapping of p21CIP1/WAF1, which reveals a unfolded conformation, consistent with NMR results (Kriwacki et al., 1997). Detection of folding pathways by partial denaturation, trapping and MS analysis of disul®de bridged intermediates has also been reported, as in the examples of EGF and M-CSF (Wilkins et al., 1993; Chang et al., 1995). Another approach is analysis of the mass/charge envelope of a protein, where the number of charges that a protein can acquire during ESI can be altered by di€erential exposure of charged residues to solvent. For example, change in the envelope upon metal binding is ascribed to increased negative charge following deprotonation of liganded residues, as observed in measurements of Ca2+ binding to calbindin (Veenstra et al., 1997) or Zn2+ binding to GAL4 (Gadhavi, 1997). Shifted charge distributions can also re¯ect conformational heterogeneity, as in the case of calmodulin, where multiple Ca2+ or Mg2+ binding states were distinguished, corresponding to high and low anity conformations (La®tte et al., 1995). Several techniques couple hydrogen/deuterium exchange with ESI-MS in which intact proteins are incubated in solvent D2O, leading to mass increases of +1 Da with each replacement of a proton by a deuteron. In one technique, intact protein masses are measured, revealing distinct conformational species in the form of molecule populations with di€ering mass distributions and peak widths. Extent and kinetics of unfolding induced with denaturants or temperature can then be measured by changes in such populations (Miranker et al., 1996; Chung et al., 1997). So far these experiments have been used to study classic model systems for spontaneous and chaperone-assisted folding as well as ribosome subunit interactions (Benjamin et al., 1998), and have not been applied to signaling molecules. In order to increase the spatial resolution of this approach, the deuterated protein can be rapidly proteolyzed and the peptides analyzed by LC/MS (Smith, 1998). Because incorporation of deuterium adds 1 Da for each amide exchanged, the rate of mass increase in individual peptides can be measured as a function of time in D2O. The mass increases can be deconvoluted into individual rate constants by least square ®t to multiple exponentials (Zhang et al., 1996) or by a maximum entropy method (Zhang et al., 1997). The results are comparable with deuterium exchange measured by NMR, but the mass spectrometric approach provides a way in which measurements on larger proteins can be achieved with lower amounts of sample. Although it is not yet clear what determines the absolute rates of exchange at a given amide, it is thought that altered exchange in local regions re¯ect ¯uctuational changes and/or mobile defects in proteins that a€ect transient solvent accessibility (Milne et al., 1998). The technique has been applied to protein folding studies and used to reveal cooperative unfolding between di€erent backbone segments in the hck-SH3 domain (Deng and Smith, 1998). In a di€erent application, the method has been used to probe di€erences between inactive and active forms of MAP kinase kinase-1, showing enhanced exchange rates upon activation, ascribed to increased backbone ¯exibility within the ATP binding domain (Resing and Ahn, 1998). Conformational changes in recoverin have also been measured by this method, showing that myristoylation stabilizes protein dynamics, and calcium binding stabilizes interactions surrounding the metal binding site (Neubert et al., 1997). Thus, mass spectrometry provides a means of monitoring aspects of solution dynamics of proteins that are currently dicult to document by other means.

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6. Conclusion It is often true in science that methodology drives discovery, and this is exempli®ed by the speed with which new developments in mass spectrometry have revolutionized analytical biochemistry. The wide-spread use of on-line search engines and availability of extensive data bases is converting science into a world community. A factor that deserves mentioning is the impact of on-line discussion groups that rapidly disseminate information about improved methodologies, in particular groups which focus on speci®c areas of signaling, for example the protein kinase discussion group fostered by the Protein Kinase Resource (http://www.sdsc.edu/ kinases/pk_home.html). As individuals create web sites that link with others, students gain access to the scienti®c vision of many mentors, and the signaling ®eld itself takes on some of the characteristics of a signaling network in an organism. Recent innovations in mass spectrometry have provided ways to analyze biomolecules with unprecedented sensitivity and accuracy, enabling investigators to address new aspects of signaling that, due to limitations in technology, were once unimaginable.

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