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The technique of hydrogen-deuterium exchange coupled to mass spectrometry (HDX-MS) has been applied to a mesophilic (E. coli) dihydrofolate reductase under conditions that allow direct comparison to a thermophilic (B. stearothermophilus) ortholog, Ec-DHFR and Bs-DHFR, respectively. The analysis of hydrogen-deuterium exchange patterns within proteolytically derived peptides allows spatial resolution, while requiring a series of controls to compare orthologous proteins with only ca. 40% sequence identity. These controls include the determination of primary structure effects on intrinsic rate constants for HDX as well as the use of existing 3-dimensional structures to evaluate the distance of each backbone amide hydrogen to the protein surface. Only a single peptide from the Ec-DHFR is found to be substantially more flexible than the Bs-DHFR at 25 °C in a region located within the protein interior at the intersection of the cofactor and substrate-binding sites. The surrounding regions of the enzyme are either unchanged or more flexible in the thermophilic DHFR from B. stearothermophilus. The region with increased flexibility in Ec-DHFR corresponds to one of two regions previously proposed to control the enthalpic barrier for hydride transfer in Bs-DHFR [Oyeyemi et al. (2010) Proc. Natl. Acad. Sci. U.S.A. 107, 10074].

The unambiguous assignment of tandem mass spectra (MS/MS) to peptide sequences remains a key unsolved problem in proteomics. Spectral library search strategies have emerged as a promising alternative for peptide identification, in which MS/MS spectra are directly compared against a reference library of confidently assigned spectra. Two problems relate to library size. First, reference spectral libraries are limited to rediscovery of previously identified peptides and are not applicable to new peptides, because of their incomplete coverage of the human proteome. Second, problems arise when searching a spectral library the size of the entire human proteome. We observed that traditional dot product scoring methods do not scale well with spectral library size, showing reduction in sensitivity when library size is increased. We show that this problem can be addressed by optimizing scoring metrics for spectrum-to-spectrum searches with large spectral libraries. MS/MS spectra for the 1.3 million predicted tryptic peptides in the human proteome are simulated using a kinetic fragmentation model (MassAnalyzer version2.1) to create a proteome-wide simulated spectral library. Searches of the simulated library increase MS/MS assignments by 24% compared with Mascot, when using probabilistic and rank based scoring methods. The proteome-wide coverage of the simulated library leads to 11% increase in unique peptide assignments, compared with parallel searches of a reference spectral library. Further improvement is attained when reference spectra and simulated spectra are combined into a hybrid spectral library, yielding 52% increased MS/MS assignments compared with Mascot searches. Our study demonstrates the advantages of using probabilistic and rank based scores to improve performance of spectrum-to-spectrum search strategies.

When analyzing proteins in complex samples using tandem mass spectrometry of peptides generated by proteolysis, the inference of proteins can be ambiguous, even with well-validated peptides. Unresolved questions include whether to show all possible proteins vs a minimal list, what to do when proteins are inferred ambiguously, and how to quantify peptides that bridge multiple proteins, each with distinguishing evidence. Here we describe IsoformResolver, a peptide-centric protein inference algorithm that clusters proteins in two ways, one based on peptides experimentally identified from MS/MS spectra, and the other based on peptides derived from an in silico digest of the protein database. MS/MS-derived protein groups report minimal list proteins in the context of all possible proteins, without redundantly listing peptides. In silico-derived protein groups pull together functionally related proteins, providing stable identifiers. The peptide-centric grouping strategy used by IsoformResolver allows proteins to be displayed together when they share peptides in common, providing a comprehensive yet concise way to organize protein profiles. It also summarizes information on spectral counts and is especially useful for comparing results from multiple LC-MS/MS experiments. Finally, we examine the relatedness of proteins within IsoformResolver groups and compare its performance to other protein inference software.

Hydrogen/deuterium exchange measurements by mass spectrometry (HX-MS) can be used to report localized conformational mobility within folded proteins, where exchange predominantly occurs through low energy fluctuations in structure, allowing transient solvent exposure. Changes in conformational mobility may impact protein function, even in cases where structural changes are unobservable. Previous studies of the MAP kinase, ERK2, revealed increases in HX upon activation occured at the hinge between conserved N- and C-terminal domains, which could be ascribed to enhanced backbone flexibility. This implied that kinase activation modulates interdomain closure, and was supported by evidence for two modes of nucleotide binding that were consistent with closed vs open conformations in active vs inactive forms of ERK2, respectively. Thus, phosphorylation of ERK2 releases constraints to interdomain closure, by modulating hinge flexibility. In this study, we examined ERK1, which shares 90% sequence identity with ERK2. HX-MS measurements of ERK1 showed similarities with ERK2 in overall deuteration, consistent with their similar tertiary structures. However, the patterns of HX that were altered upon activation of ERK1 differed from those in ERK2. In particular, alterations in HX at the hinge region upon activation of ERK2 did not occur in ERK1, suggesting that the two enzymes differ with respect to their regulation of hinge mobility and interdomain closure. In agreement, HX-MS measurements of nucleotide binding suggested revealed domain closure in both inactive and active forms of ERK1. We conclude that although ERK1 and ERK2 are closely related with respect to primary sequence and tertiary structure, they utilize distinct mechanisms for controlling enzyme function through interdomain interactions.

A complicating factor for protein identification within complex mixtures by LC/MS/MS is the problem of "chimera" spectra, where two or more precursor ions with similar mass and retention time are co-sequenced by MS/MS. Chimera spectra show reduced scores due to unidentifiable fragment ions derived from contaminating parents. However, the extent of chimeras in LC/MS/MS data sets and their impact on protein identification workflows are incompletely understood. We report ChimeraCounter, a software program which detects chimeras in data sets collected on an Orbitrap/LTQ instrument. Evaluation of synthetic chimeras created from pairs of well-defined peptide MS/MS spectra reveal that chimeras reduce database search scores most significantly when contaminating fragment ion intensities exceed 20% of the targeted fragment ion intensities. In large-scale data sets, the identification rate for chimera MS/MS is 2-fold lower compared to nonchimera spectra. Importantly, this occurs in a manner which depends not on absolute precursor ion intensity, but on intensity relative to the median precursor intensity distribution. We further show that chimeras reduce the number of accepted peptide identifications by increasing false negatives while showing little increase in false positives. The results provide a framework for identifying chimeras and characterizing their contribution to the poorly understood false negative class of MS/MS.

We report hydrogen deuterium exchange by mass spectrometry (HDX-MS) as a function of temperature in a thermophilic dihydrofolate reductase from Bacillus stearothermophilus (Bs-DHFR). Protein stability, probed with circular dichroism, established an accessible temperature range of 10 degrees C to 55 degrees C for the interrogation of HDX-MS. Although both the rate and extent of HDX are sensitive to temperature, the majority of peptides showed rapid kinetics of exchange, allowing us to focus on plateau values for the maximal extent of exchange at each temperature. Arrhenius plots of the ratio of hydrogens exchanged at 5 h normalized to the number of exchangeable hydrogens vs. 1/T provides an estimate for the apparent enthalpic change of local unfolding, DeltaH degrees (unf(avg)). Most regions in the enzyme show DeltaH degrees (unf(avg)) < or = 2.0 kcal/mol, close to the value of kT; by contrast, significantly elevated values for DeltaH degrees (unf(avg)) are observed in regions within the core of protein that contain the cofactor and substrate-binding sites. Our technique introduces a new strategy for probing the temperature dependence of local protein unfolding within native proteins. These findings are discussed in the context of the demonstrated role for nuclear tunneling in hydride transfer from NADPH to dihydrofolate, and relate the observed enthalpic changes to two classes of motion, preorganization and reorganization, that have been proposed to control the efficiency of hydrogenic wave function overlap. Our findings suggest that the enthalpic contribution to the heavy atom environmental reorganizations controlling the hydrogenic wave function overlap will be dominated by regions of the protein proximal to the bound cofactor and substrate.

In this issue of Molecular Cell, Hoshi et al. (2010) report two examples in which small molecule inhibitors are rendered ineffective when their kinase targets are involved in protein-protein interactions, highlighting differences between in vivo and in vitro inhibition kinetics., (Copyright 2010 Elsevier Inc. All rights reserved.)

Hydrogen exchange mass spectrometry (HX-MS) is an experimental technique that can be used to -examine solvent accessibility and conformational mobility in biological macromolecules. This chapter summarizes studies using HX-MS to examine the regulation of conformation, protein mobility, and ligand binding to MAP kinases. We describe the planning and design of HX-MS experiments, strategies for data analysis and interpretation, and available software.

RhoA controls changes in cell morphology and invasion associated with cancer phenotypes. Cell lines derived from melanoma tumors at varying stages revealed that RhoA is selectively activated in cells of metastatic origin. We describe a functional proteomics strategy to identify proteins regulated by RhoA and report a previously uncharacterized human protein, named "mediator of RhoA-dependent invasion (MRDI)," that is induced in metastatic cells by constitutive RhoA activation and promotes cell invasion. In human melanomas, MRDI localization correlated with stage, showing nuclear localization in nevi and early stage tumors and cytoplasmic localization with plasma membrane accentuation in late stage tumors. Consistent with its role in promoting cell invasion, MRDI localized to cell protrusions and leading edge membranes in cultured cells and was required for cell motility, tyrosine phosphorylation of focal adhesion kinase, and modulation of actin stress fibers. Unexpectedly MRDI had enzymatic function as an isomerase that converts the S-adenosylmethionine catabolite 5-methylribose 1-phosphate into 5-methylribulose 1-phosphate. The enzymatic function of MRDI was required for methionine salvage from S-adenosylmethionine but distinct from its function in cell invasion. Thus, mechanisms used by signal transduction pathways to control cell movement have evolved from proteins with ancient function in amino acid metabolism.

Human melanomas show oncogenic B-Raf mutations, which activate the B-Raf/MKK/ERK cascade. We screened microarrays to identify cellular targets of this pathway, and found that genes upregulated by B-Raf/MKK/ERK showed highest association with cell-cycle regulators, whereas genes downregulated were most highly associated with axon guidance genes, including plexin-semaphorin family members. Plexin B1 was strongly inhibited by mitogen-activated protein kinase signaling in melanoma cells and melanocytes. In primary melanoma cells, plexin B1 blocked tumorigenesis as measured by growth of colonies in soft agar, spheroids in extracellular matrix and xenograft tumors. Tumor suppression depended on residues in the C-terminal domain of plexin B1, which mediate receptor GTPase activating protein activity, and also correlated with AKT inhibition. Interestingly, the inhibitory response to plexin B1 was reduced or absent in cells from a matched metastatic tumor, suggesting that changes occur in metastatic cells which bypass the tumor-suppressor mechanisms. Plexin B1 also inhibited cell migration, but this was seen in metastatic cells and not in matched primary cells. Thus, plexin B1 has tumor-suppressor function in early-stage cells, although suppressing migration in late-stage cells. Our findings suggest that B-Raf/MKK/ERK provides a permissive environment for melanoma genesis by modulating plexin B1.

Melanoma and other cancers harbor oncogenic mutations in the protein kinase B-Raf, which leads to constitutive activation and dysregulation of MAP kinase signaling. In order to elucidate molecular determinants responsible for B-Raf control of cancer phenotypes, we present a method for phosphoprotein profiling, using negative ionization mass spectrometry to detect phosphopeptides based on their fragment ion signature caused by release of PO(3)(-). The method provides an alternative strategy for phosphoproteomics, circumventing affinity enrichment of phosphopeptides and isotopic labeling of samples. Ninety phosphorylation events were regulated by oncogenic B-Raf signaling, based on their responses to treating melanoma cells with MKK1/2 inhibitor. Regulated phosphoproteins included known signaling effectors and cytoskeletal regulators. We investigated MINERVA/FAM129B, a target belonging to a protein family with unknown category and function, and established the importance of this protein and its MAP kinase-dependent phosphorylation in controlling melanoma cell invasion into three-dimensional collagen matrix.

Identifying peptides from mass spectrometric fragmentation data (MS/MS spectra) using search strategies that map protein sequences to spectra is computationally expensive. An alternative strategy uses direct spectrum-to-spectrum matching against a reference library of previously observed MS/MS that has the advantage of evaluating matches using fragment ion intensities and other ion types than the simple set normally used. However, this approach is limited by the small sizes of the available peptide MS/MS libraries and the inability to evaluate the rate of false assignments. In this study, we observed good performance of simulated spectra generated by the kinetic model implemented in MassAnalyzer (Zhang, Z. (2004) Prediction of low-energy collision-induced dissociation spectra of peptides. Anal. Chem. 76, 3908-3922; Zhang, Z. (2005) Prediction of low-energy collision-induced dissociation spectra of peptides with three or more charges. Anal. Chem. 77, 6364-6373) as a substitute for the reference libraries used by the spectrum-to-spectrum search programs X!Hunter and BiblioSpec and similar results in comparison with the spectrum-to-sequence program Mascot. We also demonstrate the use of simulated spectra for searching against decoy sequences to estimate false discovery rates. Although we found lower score discrimination with spectrum-to-spectrum searches than with Mascot, particularly for higher charge forms, comparable peptide assignments with low false discovery rate were achieved by examining consensus between X!Hunter and Mascot, filtering results by mass accuracy, and ignoring score thresholds. Protein identification results are comparable to those achieved when evaluating consensus between Sequest and Mascot. Run times with large scale data sets using X!Hunter with the simulated spectral library are 7 times faster than Mascot and 80 times faster than Sequest with the human International Protein Index (IPI) database. We conclude that simulated spectral libraries greatly expand the search space available for spectrum-to-spectrum searching while enabling principled analyses and that the approach can be used in consensus strategies for large scale studies while reducing search times.

MAPKs are key components of cell signaling pathways with a unique activation mechanism: i.e. dual phosphorylation of neighboring threonine and tyrosine residues. The ERK enzymes form a subfamily of MAPKs involved in proliferation, differentiation, development, learning, and memory. The exact role of each Erk molecule in these processes is not clear. An efficient strategy for addressing this question is to activate individually each molecule, for example, by expressing intrinsically active variants of them. However, such molecules were not produced so far. Here, we report on the isolation, via a specifically designed genetic screen, of six variants (each carries a point mutation) of the yeast MAPK Mpk1/Erk that are active, independent of upstream phosphorylation. One of the activating mutations, R68S, occurred in a residue conserved in the mammalian Erk1 (Arg-84) and Erk2 (Arg-65) and in the Drosophila ERK Rolled (Arg-80). Replacing this conserved Arg with Ser rendered these MAPKs intrinsically active to very high levels when tested in vitro as recombinant proteins. Combination of the Arg to Ser mutation with the sevenmaker mutation (producing Erk2(R65S+D319N) and Rolled(R80S+D334N)) resulted in even higher activity (45 and 70%, respectively, in reference to fully active dually phosphorylated Erk2 or Rolled). Erk2(R65S) and Erk2(R65S+D319N) were found to be spontaneously active also when expressed in human HEK293 cells. We further revealed the mechanism of action of the mutants and show that it involves acquisition of autophosphorylation activity. Thus, a first generation of Erk molecules that are spontaneously active in vitro and in vivo has been obtained.

Hydrogen-deuterium exchange measurements represent a powerful approach to investigating changes in conformation and conformational mobility in proteins. Here, we examine p38alpha MAP kinase (MAPK) by hydrogen-exchange (HX) mass spectrometry to determine whether changes in conformational mobility may be induced by kinase phosphorylation and activation. Factors influencing sequence coverage in the HX mass spectrometry experiment, which show that varying sampling depths, instruments, and peptide search strategies yield the highest coverage of exchangeable amides, are examined. Patterns of regional deuteration in p38alpha are consistent with tertiary structure and similar to deuteration patterns previously determined for extracellular-signal-regulated kinase (ERK) 2, indicating that MAPKs are conserved with respect to the extent of local amide HX. Activation of p38alpha alters HX in five regions, which are interpreted by comparing X-ray structures of unphosphorylated p38alpha and X-ray structures of phosphorylated p38gamma. Conformational differences account for altered HX within the activation lip, the P+1 site, and the active site. In contrast, HX alterations are ascribed to activation-induced effects on conformational mobility, within substrate-docking sites (alphaF-alphaG, beta7-beta8), the C-terminal core (alphaE), and the N-terminal core region (beta4-beta5, alphaL16, alphaC). Activation also decreases HX in a 3-10 helix at the C-terminal extension of p38alpha. Although this helix in ERK2 forms a dimerization interface that becomes protected from HX upon activation, analytical ultracentrifugation shows that this does not occur in p38alpha because both unphosphorylated and diphosphorylated forms are monomeric. Finally, HX patterns in monophosphorylated p38alpha are similar to those in unphosphorylated kinase, indicating that the major activation lip remodeling events occur only after diphosphorylation. Importantly, patterns of activation-induced HX show differences between p38alpha and ERK2 despite their similarities in overall deuteration, suggesting that although MAPKs are closely related with respect to primary sequence and tertiary structure, they have distinct mechanisms for dynamic control of enzyme function.

Mechanisms by which Wnt pathways integrate the organization of receptors, organelles, and cytoskeletal proteins to confer cell polarity and directional cell movement are incompletely understood. We show that acute responses to Wnt5a involve recruitment of actin, myosin IIB, Frizzled 3, and melanoma cell adhesion molecule into an intracellular structure in a melanoma cell line. In the presence of a chemokine gradient, this Wnt-mediated receptor-actin-myosin polarity (W-RAMP) structure accumulates asymmetrically at the cell periphery, where it triggers membrane contractility and nuclear movement in the direction of membrane retraction. The process requires endosome trafficking, is associated with multivesicular bodies, and is regulated by Wnt5a through the small guanosine triphosphatases Rab4 and RhoB. Thus, cell-autonomous mechanisms allow Wnt5a to control cell orientation, polarity, and directional movement in response to positional cues from chemokine gradients.

The Mps1 protein kinase is required for proper assembly of the mitotic spindle, checkpoint signaling, and several other aspects of cell growth and differentiation. Mps1 regulation is mediated by cell cycle-dependent changes in transcription and protein level. There is also a strong correlation between hyperphosphorylated mitotic forms of Mps1 and increased kinase activity. We investigated the role that autophosphorylation plays in regulating human Mps1 (hMps1) protein kinase activity. Here we report that hyperphosphorylated hMps1 forms are not the only active forms of the kinase. However, autophosphorylation of hMps1 within the activation loop is required for full activity in vitro. Mass spectrometry analysis of de novo synthesized enzyme in Escherichia coli identified autophosphorylation sites at residues Thr(675), Thr(676), and Thr(686), but phosphatase-treated and reactivated enzyme was only phosphorylated on Thr(676). Mutation of Thr(676) in hMps1 or the corresponding Thr(591) residue within yeast Mps1 reduces kinase activity in vitro. We find that overexpression of an hMps1-T676A mutation inhibits centrosome duplication in RPE1 cells. Likewise, yeast cells harboring mps1-T591A as the sole MPS1 allele are not viable. Our data strongly support the conclusion that site-specific Mps1 autophosphorylation within the activation loop is required for full activity in vitro and function in vivo.

Post-translational modifications of proteins control many biological processes, and examining their diversity is critical for understanding mechanisms of cell regulation. Mass spectrometry is a fundamental tool for detecting and mapping covalent modifications and quantifying their changes. Modern approaches have made large-scale experiments possible, screening complex mixtures of proteins for alterations in chemical modifications. By profiling protein chemistries, biologists can gain deeper insight into biological control. The aim of this review is introduce biologists to current strategies in mass spectrometry-based proteomics that are used to characterize protein post-translational modifications, noting strengths and shortcomings of various approaches.

Proteomics addresses the important goal of determining the chemistry and composition of proteins in biological samples. Mass-spectrometry-based strategies have been highly successful in identifying and profiling proteins in complex mixtures; however, although depth of sampling continues to improve, a general recognition exists that no study has yet achieved complete protein coverage in any tissue, cell type, subcellular component, or fluid. The development of new approaches for comprehensively surveying highly complex protein mixtures, distinguishing protein isoforms, quantifying changes in protein abundance between different samples, and mapping post-translational modifications are areas of active research. These will be needed to achieve the "systems-wide" protein profiling goals of defining molecular responses to cell perturbations and obtaining biomarker information for disease detection, prognosis, and responses to therapy. We review recent progress in approaching these problems and present examples of successful applications and the outlook for the future.

A major limitation in identifying peptides from complex mixtures by shotgun proteomics is the ability of search programs to accurately assign peptide sequences using mass spectrometric fragmentation spectra (MS/MS spectra). Manual analysis is used to assess borderline identifications; however, it is error-prone and time-consuming, and criteria for acceptance or rejection are not well defined. Here we report a Manual Analysis Emulator (MAE) program that evaluates results from search programs by implementing two commonly used criteria: 1) consistency of fragment ion intensities with predicted gas phase chemistry and 2) whether a high proportion of the ion intensity (proportion of ion current (PIC)) in the MS/MS spectra can be derived from the peptide sequence. To evaluate chemical plausibility, MAE utilizes similarity (Sim) scoring against theoretical spectra simulated by MassAnalyzer software (Zhang, Z. (2004) Prediction of low-energy collision-induced dissociation spectra of peptides. Anal. Chem. 76, 3908-3922) using known gas phase chemical mechanisms. The results show that Sim scores provide significantly greater discrimination between correct and incorrect search results than achieved by Sequest XCorr scoring or Mascot Mowse scoring, allowing reliable automated validation of borderline cases. To evaluate PIC, MAE simplifies the DTA text files summarizing the MS/MS spectra and applies heuristic rules to classify the fragment ions. MAE output also provides data mining functions, which are illustrated by using PIC to identify spectral chimeras, where two or more peptide ions were sequenced together, as well as cases where fragmentation chemistry is not well predicted.

The allosteric regulation of protein kinases serves as an efficient strategy for molecular communication, event coupling and interconversion between catalytic states. Recent co-crystal structures have revealed novel ways in which kinases control activity and substrate specificity following phosphorylation, dimerization, or binding to regulatory proteins, substrates and scaffolds. In addition, hydrogen exchange coupled with mass spectrometry is emerging as a complementary strategy to probe the solution behavior of kinases; recent results have shown that allosteric regulation may involve transitions in protein motions as well as structural rearrangements.

Studies of protein kinases have identified a "gatekeeper" residue, which confers selectivity for binding nucleotides and small-molecule inhibitors. We report that, in the MAP kinase ERK2, mutations at the gatekeeper residue unexpectedly lead to autoactivation due to enhanced autophosphorylation of regulatory Tyr and Thr sites within the activation lip that control kinase activity. This occurs through an intramolecular mechanism, indicating that the gatekeeper residue indirectly constrains flexibility at the activation lip, precluding access of the phosphoacceptor residues to the catalytic base. Other residues that interact with the gatekeeper site to form a hydrophobic cluster in the N-terminal domain also cause autoactivation when mutated. Hydrogen-exchange studies of a mutant within this cluster reveal perturbations in the conserved DFG motif, predicting a route for side chain connectivity from the hydrophobic cluster to the activation lip. Mutations of residues along this route support this model, explaining how information about the gatekeeper residue identity can be transmitted to the activation lip. Thus, an N-terminal hydrophobic cluster that includes the gatekeeper forms a novel structural unit, which functions to maintain the "off" state of ERK2 before cell signal activation.

Abnormal amplification of centrosomes, commonly found in human cancer, is the major cause of mitotic defects and chromosome instability in cancer cells. Like DNA, centrosomes duplicate once in each cell cycle, hence the defect in the mechanism that ensures centrosome duplication to occur once and only once in each cell cycle results in abnormal amplification of centrosomes and mitotic defects. Centrosomes are non-membranous organelles, and undergo dynamic changes in its constituents during the centrosome duplication cycle. Through a comparative mass spectrometric analysis of unduplicated and duplicated centrosomes, we identified mortalin, a member of heat shock protein family, as a protein that associates preferentially with duplicated centrosomes. Further analysis revealed that mortalin localized to centrosomes in late G1 before centrosome duplication, remained at centrosomes during S and G2, and dissociated from centrosomes during mitosis. Overexpression of mortalin overrides the p53-dependent suppression of centrosome duplication, and mortalin-driven centrosome duplication requires physical interaction between mortalin and p53. Moreover, mortalin promotes dissociation of p53 from centrosomes through physical interaction. The p53 mutant that lacks the ability to bind to mortalin remains at centrosomes, and suppresses centrosome duplication in a transactivation function-independent manner. Thus, our present findings not only identify mortalin as an upstream molecule of p53 but also provide evidence for the involvement of centrosomally localized p53 in the regulation of centrosome duplication.

Rho GTPases are signal transduction effectors that control cell motility, cell attachment, and cell shape by the control of actin polymerization and tyrosine phosphorylation. To identify cellular targets regulated by Rho GTPases, we screened global protein responses to Rac1, Cdc42, and RhoA activation by two-dimensional gel electrophoresis and mass spectrometry. A total of 22 targets were identified of which 19 had never been previously linked to Rho GTPase pathways, providing novel insight into pathway function. One novel target of RhoA was protein-tyrosine phosphatase 1B (PTP1B), which catalyzes dephosphorylation of key signaling molecules in response to activation of diverse pathways. Subsequent analysis demonstrated that RhoA enhances post-translational modification of PTP1B, inactivates phosphotyrosine phosphatase activity, and up-regulates tyrosine phosphorylation of p130Cas, a key mediator of focal adhesion turnover and cell migration. Thus, protein profiling reveals a novel role for PTP1B as a mediator of RhoA-dependent phosphorylation of p130Cas.

Although the importance of the extracellular signal-regulated kinase (ERK) pathway in regulating the transition from G1 to S has been extensively studied, its role during the G2/M transition is less well understood. Previous reports have shown that inhibition of the ERK pathway in mammalian cells delays entry as well as progression through mitosis, suggesting the existence of molecular targets of this pathway in M phase. In this report we employed 2-DE and MS to survey proteins and PTMs in the presence versus absence of MKK1/2 inhibitor. Targets of the ERK pathway in G2/M were identified as elongation factor 2 (EF2) and nuclear matrix protein, 55 kDa (Nmt55). Phosphorylation of each protein increased under conditions of ERK pathway inhibition, suggesting indirect control of these targets; regulation of EF2 was ascribed to phosphorylation and inactivation of upstream EF2 kinase, whereas regulation of Nmt55 was ascribed to a delay in normal mitotic phosphorylation and dephosphorylation. 2-DE Western blots probed using anti-phospho-Thr-Pro antibody demonstrated that the effect of ERK inhibition is not to delay the onset of phosphorylation controlled by cdc2 and other mitotic kinases, but rather to regulate a small subset of targets in M phase in a nonoverlapping manner with cdc2.

ERK5 is a recently characterized MAPK, which is most similar to the well studied ERK1/2 subfamily but uses distinct mechanisms to elicit responses. To understand the specificity of signaling through ERK5 versus ERK1/2, we examined global gene expression changes in response to each pathway. Microarray measurements in retinal pigment epithelial cells revealed 36 genes regulated by ERK5, all which were novel targets for this pathway. 39 genes were regulated by ERK1/2, which included 11 known genes. Of these genes, 19 were regulated by both pathways. Inspection of the 17 genes uniquely regulated by ERK5 revealed that 14 genes (82%) were previously associated with hypoxia via regulation by HIF-1. In contrast, 16 genes (84%) regulated by either ERK5 or ERK1/2 were implicated in hypoxia, most through mechanisms independent of HIF-1. Of the 20 genes regulated by ERK1/2, only 9 were implicated in hypoxia and were not well characterized hypoxia targets. Thus, unlike ERK5, a mechanistic link between ERK1/2 and HIF-1/HRE could not be established on the basis of gene regulation. Activation of both pathways enhanced transcription from a hypoxia-response element and increased HIF-1alpha protein expression. In contrast, ERK5 but not ERK1/2 elevated transcription through GAL4-HIF-1. Most interestingly, ERK5 is not significantly activated by hypoxia in retinal pigment epithelial cells, indicating that ERK5 regulation of these genes is relevant in normoxia rather than hypoxia. Thus, ERK5 and ERK1/2 differ in their mechanisms of gene regulation, and indicate that ERK5 may control hypoxia-responsive genes by a mechanism independent of HIF-1alpha expression control.

Integrating biological information from different sources to understand cellular processes is an important problem in systems biology. We use data from mRNA expression arrays and chemical kinetics to formulate a metabolic model relevant to K562 erythroleukemia cells. MAP kinase pathway activation alters the expression of metabolic enzymes in K562 cells. Our array data show changes in expression of lactate dehydrogenase (LDH) isoforms after treatment with phorbol 12-myristate 13-acetate (PMA), which activates MAP kinase signaling. We model the change in lactate production which occurs when the MAP kinase pathway is activated, using a non-equilibrium, chemical-kinetic model of homolactic fermentation. In particular, we examine the role of LDH isoforms, which catalyse the conversion of pyruvate to lactate. Changes in the isoform ratio are not the primary determinant of the production of lactate. Rather, the total concentration of LDH controls the lactate concentration.

Overexpression of the small GTPase, RhoC, in various human cancers has been correlated with high metastatic ability and poor prognosis. Rho-kinase (ROCK) is an important effector of Rho GTPases. The oncogenic serine/threonine kinase Akt (also known as PKB) is a downstream effector of phosphatidylinositol-3 kinase (PI3K). Akt activation contributes to the neoplastic phenotype by promoting cell cycle progression, increasing antiapoptotic functions, and enhancing tumor cell invasion. Rho signaling via ROCK has been previously shown either to activate or to downregulate PI3K/Akt. Using a human radial growth phase melanoma cell line, WM35, we have established stable transfectants that overexpress RhoC (called WM35RhoC). We found that overexpression of RhoC increased phosphorylated-Akt (Ser473/474/472, pAkt) expression and promoted cell invasion. Inhibition of RhoC with C3 transferase downregulated pAkt expression and decreased cell invasion in these cells. In addition, inhibition of PI3K, Akt, or ROCK partially decreased invasion. Further, inhibition of PI3K but not ROCK decreased the pAkt level. These results suggest that RhoC promotes invasion in part via activation of a PI3K/Akt pathway, in a manner independent of ROCK signaling. We propose that RhoC promotes melanoma progression via separate mechanisms that regulate the PI3K/Akt pathway and the ROCK signaling pathway.

An important strategy for "shotgun proteomics" profiling involves solution proteolysis of proteins, followed by peptide separation using multidimensional liquid chromatography and automated sequencing by mass spectrometry (LC-MS/MS). Several protocols for extracting and handling membrane proteins for shotgun proteomics experiments have been reported, but few direct comparisons of different protocols have been reported. We compare four methods for preparing membrane proteins from human cells, using acid labile surfactants (ALS), urea, and mixed organic-aqueous solvents. These methods were compared with respect to their efficiency of protein solubilization and proteolysis, peptide and protein recovery, membrane protein enrichment, and peptide coverage of transmembrane proteins. Overall, approximately 50-60% of proteins recovered were membrane-associated, identified from Gene Ontology annotations and transmembrane prediction software. Samples extracted with ALS, extracted with urea followed by dilution, or extracted with urea followed by desalting yielded comparable peptide recoveries and sequence coverage of transmembrane proteins. In contrast, suboptimal proteolysis was observed with organic solvent. Urea extraction followed by desalting may be a particularly useful approach, as it is less costly than ALS and yields satisfactory protein denaturation and proteolysis under conditions that minimize reactivity with urea-derived cyanate. Spectral counting was used to compare datasets of proteins from membrane samples with those of soluble proteins from K562 cells, and to estimate fold differences in protein abundances. Proteins most highly abundant in the membrane samples showed enrichment of integral membrane protein identifications, consistent with their isolation by differential centrifugation.

Correct identification of a peptide sequence from MS/MS data is still a challenging research problem, particularly in proteomic analyses of higher eukaryotes where protein databases are large. The scoring methods of search programs often generate cases where incorrect peptide sequences score higher than correct peptide sequences (referred to as distraction). Because smaller databases yield less distraction and better discrimination between correct and incorrect assignments, we developed a method for editing a peptide-centric database (PC-DB) to remove unlikely sequences and strategies for enabling search programs to utilize this peptide database. Rules for unlikely missed cleavage and nontryptic proteolysis products were identified by data mining 11 849 high-confidence peptide assignments. We also evaluated ion exchange chromatographic behavior as an editing criterion to generate subset databases. When used to search a well-annotated test data set of MS/MS spectra, we found no loss of critical information using PC-DBs, validating the methods for generating and searching against the databases. On the other hand, improved confidence in peptide assignments was achieved for tryptic peptides, measured by changes in DeltaCN and RSP. Decreased distraction was also achieved, consistent with the 3-9-fold decrease in database size. Data mining identified a major class of common nonspecific proteolytic products corresponding to leucine aminopeptidase (LAP) cleavages. Large improvements in identifying LAP products were achieved using the PC-DB approach when compared with conventional searches against protein databases. These results demonstrate that peptide properties can be used to reduce database size, yielding improved accuracy and information capture due to reduced distraction, but with little loss of information compared to conventional protein database searches.

Structural and kinetic studies have provided extensive information about the molecular mechanisms of kinase activation by phosphorylation. However, it is still unclear how changes in protein dynamics and flexibility contribute to catalytic function. Mass spectrometry was used to probe changes in hydrogen/deuterium exchange in the MAP kinase, ERK2, in the presence and absence of the ATP analogue, AMP-PNP. In both active and inactive forms of ERK2, protection from hydrogen exchange by AMP-PNP binding was observed within conserved ATP binding motifs in the N-terminal lobe, which are known to directly interact with nucleotide in various protein kinases. In contrast, higher protection from exchange by AMP-PNP was observed in active ERK2 compared to inactive ERK2, in a region corresponding to the conserved DFG motif, which is located in the C-terminal lobe and coordinates Mg2+ at the catalytic site. Thus, AMP-PNP binding simultaneously protects residues within the N and C terminus in the active form of ERK2, but not the inactive form. This demonstrates that ERK2 binds nucleotide in two modes, in which active ERK2 adopts a closed conformation following nucleotide binding in solution, while inactive ERK2 adopts an open conformation. The finding provides novel evidence that phosphorylation of ERK2 facilitates interdomain closure, allowing proper orientation between ATP and substrate to facilitate phosphoryl transfer.

Measurements of mass spectral peak intensities and spectral counts are promising methods for quantifying protein abundance changes in shotgun proteomic analyses. We describe Serac, software developed to evaluate the ability of each method to quantify relative changes in protein abundance. Dynamic range and linearity using a three-dimensional ion trap were tested using standard proteins spiked into a complex sample. Linearity and good agreement between observed versus expected protein ratios were obtained after normalization and background subtraction of peak area intensity measurements and correction of spectral counts to eliminate discontinuity in ratio estimates. Peak intensity values useful for protein quantitation ranged from 10(7) to 10(11) counts with no obvious saturation effect, and proteins in replicate samples showed variations of less than 2-fold within the 95% range (+/-2sigma) when >or=3 peptides/protein were shared between samples. Protein ratios were determined with high confidence from spectral counts when maximum spectral counts were >or=4 spectra/protein, and replicates showed equivalent measurements well within 95% confidence limits. In further tests, complex samples were separated by gel exclusion chromatography, quantifying changes in protein abundance between different fractions. Linear behavior of peak area intensity measurements was obtained for peptides from proteins in different fractions. Protein ratios determined by spectral counting agreed well with those determined from peak area intensity measurements, and both agreed with independent measurements based on gel staining intensities. Overall spectral counting proved to be a more sensitive method for detecting proteins that undergo changes in abundance, whereas peak area intensity measurements yielded more accurate estimates of protein ratios. Finally these methods were used to analyze differential changes in protein expression in human erythroleukemia K562 cells stimulated under conditions that promote cell differentiation by mitogen-activated protein kinase pathway activation. Protein changes identified with p<0.1 showed good correlations with parallel measurements of changes in mRNA expression.

The information from genome sequencing provides new approaches for systems-wide understanding of protein networks and cellular function. DNA microarray technologies have advanced to the point where nearly complete monitoring of gene expression is feasible in several organisms. An equally important goal is to comprehensive survey cellular proteomes and profile protein changes under different cellular states. This presents a complex analytical problem, due to the chemical variability between proteins and peptides. Here, we discuss strategies to improve accuracy and sensitivity of peptide identification, distinguish represented protein isoforms, and quantify relative changes in protein abundance.

The psychrophilic alcohol dehydrogenase (psADH) cloned from Antarctic Moraxella sp. TAE123 exhibits distinctive catalytic parameters in relation to the homologous thermophilic alcohol dehydrogenase (htADH) from Bacillus stearothermophilus LLD-R. Amide hydrogen-deuterium (H/D) exchange studies using Fourier-transformed infrared (FTIR) spectroscopy and mass spectrometry (MS) were conducted to investigate whether the differences are caused by variation in either global or regional protein flexibility. The FTIR H/D exchange study suggested that psADH does not share similar global flexibility with htADH at their physiologically relevant temperatures as has been predicted by the "corresponding state" hypothesis. However, the MS H/D exchange study revealed a more complicated picture concerning the flexibility of the two homologous enzymes. Analysis of the deuteration and exchange rates of protein-derived peptides suggested that only some functionally important regions in psADH that are involved in substrate and cofactor binding exhibit greater flexibility compared to htADH at low temperature (10 degrees C). These observations strongly suggest that variable conformational flexibility between the two protein forms is a local phenomenon, and that global H/D exchange measurement by FTIR can be misleading and should be used with discretion. These results are supportive of the idea that functionally important local flexibility can be uncoupled from global thermal stability. The structural factors underlying the differences in local protein flexibility and catalysis between htADH and psADH are discussed in conjunction with results from crystallographic and fluorescence spectroscopy studies.

The information from genome sequencing provides a new framework for a systems-wide understanding of protein networks and cellular function. Whereas microarray technologies provide information about global gene expression within cells, complementary proteomic strategies monitor expression of proteins and their posttranslational modifications. Improved technologies that have emerged for comprehensive and high-throughput protein analysis yield novel insights into cell regulation.

Duplication of the Saccharomyces cerevisiae spindle pole body (SPB) once per cell cycle is essential for bipolar spindle formation and accurate chromosome segregation during mitosis. We have investigated the role that the major yeast cyclin-dependent kinase Cdc28/Cdk1 plays in assembly of a core SPB component, Spc42, to better understand how SPB duplication is coordinated with cell cycle progression. Cdc28 is required for SPB duplication and Spc42 assembly, and we found that Cdc28 directly phosphorylates Spc42 to promote its assembly into the SPB. The Mps1 kinase, previously shown to regulate Spc42 phosphorylation and assembly, is also a Cdc28 substrate, and Cdc28 phosphorylation of Mps1 is needed to maintain wild-type levels of Mps1 in cells. Analysis of nonphosphorylatable mutants in SPC42 and MPS1 indicates that direct Spc42 phosphorylation and indirect regulation of Spc42 through Mps1 are two overlapping pathways by which Cdc28 regulates Spc42 assembly and SPB duplication during the cell cycle.

Identifying proteins in cell extracts by shotgun proteomics involves digesting the proteins, sequencing the resulting peptides by data-dependent mass spectrometry (MS/MS), and searching protein databases to identify the proteins from which the peptides are derived. Manual analysis and direct spectral comparison reveal that scores from two commonly used search programs (Sequest and Mascot) validate less than half of potentially identifiable MS/MS spectra (class positive) from shotgun analyses of the human erythroleukemia K562 cell line. Here we demonstrate increased sensitivity and accuracy using a focused search strategy along with a peptide sequence validation script that does not rely exclusively on XCorr or Mowse scores generated by Sequest or Mascot, but uses consensus between the search programs, along with chemical properties and scores describing the nature of the fragmentation spectrum (ion score and RSP). The approach yielded 4.2% false positive and 8% false negative frequencies in peptide assignments. The protein profile is then assembled from peptide assignments using a novel peptide-centric protein nomenclature that more accurately reports protein variants that contain identical peptide sequences. An Isoform Resolver algorithm ensures that the protein count is not inflated by variants in the protein database, eliminating approximately 25% of redundant proteins. Analysis of soluble proteins from a human K562 cells identified 5130 unique proteins, with approximately 100 false positive protein assignments.

Temperature-dependent hydrogen-deuterium (H/D) exchange of the thermophilic alcohol dehydrogenase (htADH) has been studied by using liquid chromatography-coupled mass spectrometry. Analysis of the changes in H/D exchange patterns for the protein-derived peptides suggests that some regions of htADH are in a rigid conformational substate at reduced temperatures with limited cooperative protein motion. The enzyme undergoes two discrete transitions at approximately 30 and 45 degrees C to attain a more dynamic conformational substate. Four of the five peptides exhibiting the transition above 40 degrees C are in direct contact with the cofactor, and the NAD(+)-binding affinity is also altered in this temperature range, implicating a change in the mobility of the cofactor-binding domain >45 degrees C. By contrast, the five peptides exhibiting the transition at 30 degrees C reside in the substrate-binding domain. This transition coincides with a change in the activation energy of k(cat) for hydride transfer, leading to a linear correlation between k(cat) and the weighted average exchange rate constant k(HX(WA)) for the five peptides. These observations indicate a direct coupling between hydride transfer and protein mobility in htADH, and that an increased mobility is at least partially responsible for the reduced E(act) at high temperature. The data provide support for the hypothesis that protein dynamics play a key role in controlling hydrogen tunneling at enzyme active sites.

Extracellular signal-regulated kinase (ERK) facilitates cell cycle progression in most mammalian cells, but in certain cell types prolonged signaling through this pathway promotes differentiation and lineage-specific gene expression through mechanisms that are poorly understood. Here, we characterize the transcriptional regulation of platelet GPIIb integrin (CD41) by ERK during megakaryocyte differentiation. ERK-dependent transactivation involves the proximal promoter of GPIIb within 114 bp upstream of the transcriptional start site. GATA, Ets, and Sp1 consensus sequences within this region are each necessary and function combinatorially in ERK-activated transcription. MafB/Kreisler is induced in response to ERK and synergizes with GATA and Ets to enhance transcription from the proximal promoter. The requirement for MafB in promoter regulation is demonstrated by inhibition of transactivation following dominant-negative or antisense suppression of MafB function. Thus, ERK promotes megakaryocyte differentiation by coordinate regulation of nuclear factors that synergize in GPIIb promoter regulation. These results establish a novel role for MafB as a regulator of ERK-induced gene expression.

Protein interactions between MAP kinases and substrates, activators, and scaffolding proteins are regulated by docking site motifs, one containing basic residues proximal to Leu-X-Leu (DEJL) and a second containing Phe-X-Phe (DEF). Hydrogen exchange mass spectrometry was used to identify regions in MAP kinases protected from solvent by docking motif interactions. Protection by DEJL peptide binding was observed in loops spanning beta7-beta8 and alphaD-alphaE in p38alpha and ERK2. In contrast, protection by DEF binding to ERK2 revealed a distinct hydrophobic pocket for Phe-X-Phe binding formed between the P+1 site, alphaF helix, and the MAP kinase insert. In inactive ERK2, this pocket is occluded by intramolecular interactions with residues in the activation lip. In vitro assays confirm the dependence of Elk1 and nucleoporin binding on ERK2 phosphorylation, and provide a structural basis for preferential involvement of active ERK in substrate binding and nuclear pore protein interactions.

Mitogen-activated protein kinases are regulated by occupancy at two phosphorylation sites near the active site cleft. Previous studies using hydrogen exchange to investigate the canonical mitogen-activated protein kinase, extracellular signal-regulated protein kinase-2, have shown that phosphorylation alters backbone conformational mobility >10 A distal to the site of phosphorylation, including decreased mobility within amino acids 102-105 and increased mobility within 108-109. To further describe changes after enzyme activation, site-directed spin labeling at amino acids 101, 105-109, 111, 112 and electron paramagnetic resonance spectroscopy were used to investigate this region. The anisotropic hyperfine splitting of the spin labels in glassy samples was unchanged by phosphorylation, consistent with previous crystallographic studies that indicate no structural change in this region. At positions 101, 111, and 112, the mobility of the spin label was unchanged by diphosphorylation, consistent with little or no conformational change. However, diphosphorylation caused small but significant changes in rotational diffusion rates at positions 105-108 and altered proportions of probe in a motionally constrained state at positions 105, 107, and 109. Thus, electron paramagnetic resonance indicates reproducible changes in nanosecond side-chain mobilities at specific residues within the interdomain region, far from the site of phosphorylation and conformational change.

Two-dimensional electrophoresis (2-DE) is a powerful technique to differentially display patterns of protein expression and posttranslational modifications, providing a good strategy to monitor molecular responses induced by the activation or inactivation of specific signaling pathways. In this chapter, optimized protocols for 2-DE using extracts from tissue culture are provided. Protocols for in-gel digestion of gel-resolved proteins, which allow protein identification by mass spectrometry are also discussed.

Functional proteomics provides a powerful approach to screen for alterations in protein expression and posttranslational modifications under conditions of human disease. In this study, we use protein screening to examine markers of melanoma progression, by profiling melanocyte versus melanoma cell lines using two-dimensional electrophoresis and mass spectrometry. Eight candidate markers were identified as differentially regulated in transformed cells. In particular, hepatoma-derived growth factor (HDGF) and nucleophosmin B23 were strongly correlated with melanoma. Nucleophosmin B23 is a nucleolar and centrosome-associated protein, which has been implicated as a target for cyclin E/cyclin-dependent kinase 2 (CDK2) in modulating centrosome duplication and cell cycle control. Western blotting of one-dimensional and two-dimensional gels showed that the form of nucleophosmin B23 that is up-regulated in melanoma represents a posttranslationally modified form, most likely reflecting enhanced phosphorylation in the tumor-derived cells. In contrast, Western analysis of HDGF demonstrated increased expression of all forms in melanoma cell lines compared with melanocytes. Immunohistochemical analysis of human tissue biopsies showed strong expression of HDGF in early and late stage melanomas and low expression in melanocytes and nontumorigenic nevi. Interestingly, biopsies of nevi showed a graded effect in which HDGF immunoreactivity was reduced in nevoid nests penetrating deep into the dermis compared with nests at the epidermal-dermal junction, suggesting that HDGF expression in nevi is dependent on epidermal cell interactions. In contrast, biopsies of melanoma showed strong expression of HDGF throughout the tumor, including cells located deeply within dermis. Thus, expression of this antigen likely reports a reduced dependence of protein expression on epidermal interactions.

Mass spectrometric analysis of proteolytically derived phosphopeptides has developed into a widespread technique for the identification of phosphorylated amino acids. Using liquid chromatography-electrospray ionization tandem mass spectrometry, 14 phosphorylation sites were identified on Xenopus laevis His6-Aurora A, a highly conserved regulator of centrosome maturation and cell division. These included seven novel phosphorylation sites, Ser-12, Thr-21, Thr-103, Ser-116, Thr-122, Tyr-155, and Thr-294, as well as the previously identified regulatory sites, Ser-53, Thr-295, and Ser-349. The identification of these novel phosphorylation sites will be important for future studies aimed at elucidating the mechanisms of Aurora A regulation by phosphorylation. Furthermore, we demonstrate that a "kinase-inactive" mutant of Aurora A, K169R, still retains 10% of activity of the wild-type enzyme in vitro along with occupancy of Thr-295 and Ser-12. However, mutation of Asp-281 to Ala completely abolishes activity of the enzyme and should therefore be used preferentially as a genuine kinase-dead construct. Because of the abundance of phosphorylated residues on His6-Aurora A, we found this protein to be an ideal tool for the characterization of immobilized metal-affinity chromatography (IMAC) as a method for phosphopeptide enrichment from complex mixtures. We present a detailed analysis of the binding and elution properties of both the phosphopeptides and unphosphorylated peptides of His6-Aurora A to Fe3+-IMAC before and after methyl esterification. Moreover, we demonstrate a significant difference in enrichment of phosphopeptides when different resins are used for Fe3+-IMAC and characterize the strengths and limitations of this methodology for the study of phosphoproteomics.

Posttranslational modification of chromatin-associated proteins, including histones and high-mobility-group (HMG) proteins, provides an important mechanism to control gene expression, genome integrity, and epigenetic inheritance. Protein mass analysis provides a rapid and unbiased approach to monitor multiple chemical modifications on individual molecules. This review describes methods for acid extraction of histones and HMG proteins, followed by separation by reverse-phase chromatography coupled to electrospray ionization mass spectrometry (LC/ESI-MS). Posttranslational modifications are detected by analysis of full-length protein masses. Confirmation of protein identity and modification state is obtained through enzymatic digestion and peptide sequencing by MS/MS. For differentially modified forms of each protein, the measured intensities are semiquantitative and allow determination of relative abundance and stoichiometry. The method simultaneously detects covalent modifications on multiple proteins and provides a facile assay for comparing chromatin modification states between different cell types and/or cellular responses.