Determination of the stoichiometry of protein complexes using liquid chromatography with fluorescence and mass spectrometric detection of fluorescently labeled proteolytic peptides

A method for the determination of the stoichiometry of protein complexes has been developed, which is based on proteolytic digestion of the complex, labeling with a fluorescent reagent, specific for amino or sulfhydryl groups, and separation by liquid chromatography with fluorescence and mass spectrometric detection. The intensity of the fluorescence signal of the labeled peptides resulting from different proteins is directly proportional to the stoichiometry of these proteins in the complex. The performance of the method was evaluated with standard peptides and proteins to ensure that accurate molar ratios can be obtained from the fluorescence chromatogram. Standard deviations of the measured molar ratio from the expected molar ratio were below 10% for both peptides and proteins. The method was finally employed for the determination of the stoichiometry of the 1:1 complex of sFc gamma RIII and hFc1. Using the described methodology, a stoichiometry of 1:1.1 was measured, which agrees well with a 1:1 complex.     

Comprehensive Analysis of Individual Variation in the Urinary Proteome Revealed Significant Gender Differences

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Highlights

• •Comprehensive analysis of inter-individual variation of normal urinary proteome.
• •Significant gender differences were observed.
• •Proteins increased in female urine are enriched in immunological pathways.
• •Estimated reference intervals of proteins as the baseline for biomarker discovery.

     

Endosomal targeting of the phosphoinositide 3-phosphatase MTMR2 is regulated by an N-terminal phosphorylation site

MTMR2 is a member of the myotubularin family of inositol lipid phosphatases, a large protein-tyrosine phosphatase subgroup that is conserved from yeast to humans. Furthermore, the peripheral neuromuscular disease Charcot-Marie Tooth disease type 4B has been attributed to mutations in the mtmr2 gene. Because the molecular mechanisms regulating MTMR2 have been poorly defined, we investigated whether reversible phosphorylation might regulate MTMR2 function. We used mass spectrometry-based methods to identify a high stoichiometry phosphorylation site on serine 58 of MTMR2. Phosphorylation at Ser(58), or a phosphomimetic S58E mutation, markedly decreased MTMR2 localization to endocytic vesicular structures. In contrast, a phosphorylation-deficient MTMR2 mutant (S58A) displayed constitutive localization to early endocytic structures. This localization pattern was accompanied by displacement of a PI(3)P-specific sensor protein and an increase in signal transduction pathways. Thus, MTMR2 phosphorylation is likely to be a critical mechanism by which MTMR2 access to its lipid substrate(s) is temporally and spatially regulated, thereby contributing to the control of downstream endosome maturation events.     

Engineering the Pattern of Protein Glycosylation Modulates the Thermostability of a GH11 Xylanase

Protein glycosylation is a common post-translational modification, the effect of which on protein conformational and stability is incompletely understood. Here we have investigated the effects of glycosylation on the thermostability of Bacillus subtilis xylanase A (XynA) expressed in Pichia pastoris. Intact mass analysis of the heterologous wild-type XynA revealed two, three, or four Hex_8–16GlcNAc₂ modifications involving asparagine residues at positions 20, 25, 141, and 181. Molecular dynamics (MD) simulations of the XynA modified with various combinations of branched Hex₉GlcNAc₂ at these positions indicated a significant contribution from protein-glycan interactions to the overall energy of the glycoproteins. The effect of glycan content and glycosylation position on protein stability was evaluated by combinatorial mutagenesis of all six potential N-glycosylation sites. The majority of glycosylated enzymes expressed in P. pastoris presented increased thermostability in comparison with their unglycosylated counterparts expressed in Escherichia coli. Steric effects of multiple glycosylation events were apparent, and glycosylation position rather than the number of glycosylation events determined increases in thermostability. The MD simulations also indicated that clustered glycan chains tended to favor less stabilizing glycan-glycan interactions, whereas more dispersed glycosylation patterns favored stabilizing protein-glycan interactions.     

Efficient Use of Exogenous Isoprenols for Protein Isoprenylation by MDA-MB-231 Cells Is Regulated Independently of the Mevalonate Pathway

Mammalian cells can use exogenous isoprenols to generate isoprenoid diphosphate substrates for protein isoprenylation, but the mechanism, efficiency, and biological importance of this process are not known. We developed mass spectrometry-based methods using chemical probes and newly synthesized stable isotope-labeled tracers to quantitate incorporation of exogenously provided farnesol, geranylgeraniol, and unnatural analogs of these isoprenols containing an aniline group into isoprenoid diphosphates and protein isoprenylcysteines by cultured human cancer cell lines. We found that at exogenous isoprenol concentrations >10 μm, this process can generate as much as 50% of the cellular isoprenoid diphosphate pool used for protein isoprenylation. Mutational activation of p53 in MDA-MB-231 breast cancer cells up-regulates the mevalonate pathway to promote tumor invasiveness. p53 silencing or pharmacological inhibition of HMG-CoA reductase in these cells decreases protein isoprenylation from endogenously synthesized isoprenoids but enhances the use of exogenous isoprenols for this purpose, indicating that this latter process is regulated independently of the mevalonate pathway. Our observations suggest unique opportunities for design of cancer cell-directed therapies and may provide insights into mechanisms underlying pleiotropic therapeutic benefits and unwanted side effects of mevalonate pathway inhibition.     

The Activity of Hyaluronan Synthase 2 Is Regulated by Dimerization and Ubiquitination

Hyaluronan is a component of the extracellular matrix, which affects tissue homeostasis. In this study, we investigated the regulatory mechanisms of one of the hyaluronan-synthesizing enzymes, HAS2. Ectopic expression of Flag- and 6myc-HAS2 in COS-1 cells followed by immunoprecipitation and immunoblotting revealed homodimers; after co-transfection with Flag-HAS3, also heterodimers were seen. Furthermore, the expressed HAS2 was ubiquitinated. We identified one acceptor site for ubiquitin on lysine residue 190. Mutation of this residue led to inactivation of the enzymatic activity of HAS2. Interestingly, K190R-mutated HAS2 formed dimers with wt HAS2 and quenched the activity of wt HAS2, thus demonstrating a functional role of the dimeric configuration.     

Multimethylation of Rickettsia OmpB Catalyzed by Lysine Methyltransferases

Methylation of rickettsial OmpB (outer membrane protein B) has been implicated in bacterial virulence. Rickettsial methyltransferases RP789 and RP027-028 are the first biochemically characterized methyltransferases to catalyze methylation of outer membrane protein (OMP). Methylation in OMP remains poorly understood. Using semiquantitative integrated liquid chromatography-tandem mass spectroscopy, we characterize methylation of (i) recombinantly expressed fragments of Rickettsia typhi OmpB exposed in vitro to trimethyltransferases of Rickettsia prowazekii RP027-028 and of R. typhi RT0101 and to monomethyltransferases of R. prowazekii RP789 and of R. typhi RT0776, and (ii) native OmpBs purified from R. typhi and R. prowazekii strains Breinl, RP22, and Madrid E. We found that in vitro trimethylation occurs at relatively specific locations in OmpB with consensus motifs, KX(G/A/V/I)N and KT(I/L/F), whereas monomethylation is pervasive throughout OmpB. Native OmpB from virulent R. typhi contains mono- and trimethyllysines at locations well correlated with methylation in recombinant OmpB catalyzed by methyltransferases in vitro. Native OmpBs from highly virulent R. prowazekii strains Breinl and RP22 contain multiple clusters of trimethyllysine in contrast to a single cluster in OmpB from mildly virulent R. typhi. Furthermore, OmpB from the avirulent strain Madrid E contains mostly monomethyllysine and no trimethyllysine. The native OmpB from Madrid E was minimally trimethylated by RT0101 or RP027-028, consistent with a processive mechanism of trimethylation. This study provides the first in-depth characterization of methylation of an OMP at the molecular level and may lead to uncovering the link between OmpB methylation and rickettsial virulence.     

Breaking the light and heavy chain linkage of human immunoglobulin G1 (IgG1) by radical reactions

We report that the production of hydrogen peroxide by radical chain reductions of molecular oxygen into water in buffers leads to hinge degradation of a human IgG1 under thermal incubation conditions. The production of the hydrogen peroxide can be accelerated by superoxide dismutase or redox active metal ions or inhibited by free radical scavengers. The hydrogen peroxide production rate correlates well with the hinge cleavage. In addition to radical reaction mechanisms described previously, new degradation pathways and products were observed. These products were determined to be generated via radical reactions initiated by electron transfer and addition to the interchain disulfide bond between Cys(215) of the light chain and Cys(225) of the heavy chain. Decomposition of the resulting disulfide bond radical anion breaks the C-S bond at the side chain of Cys, converting it into dehydroalanine and generating a sulfur radical adduct at its counterpart. The hydrolysis of the unsaturated dehydropeptides removes Cys and yields an amide at the C terminus of the new fragment. Meanwhile, the competition between the carbonyl (-C(α)ONH-) and the side chain of Cys allows an electron transfer to the α carbon, forming a new intermediate radical species (-(·)C(α)(O(-))NH-) at Cys(225). Dissociative deamidation occurs along the N-C(α) bond, resulting in backbone cleavage. Given that hydrogen peroxide is a commonly observed product of thermal stress and plays a role in mediating the unique degradation of an IgG1, strategies for improving stability of human antibody therapeutics are discussed.     

Ligand Binding to Anion-binding Exosites Regulates Conformational Properties of Thrombin

Thrombin participates in coagulation, anticoagulation, and initiation of platelet activation. To fulfill its diverse roles and maintain hemostasis, this serine protease is regulated via the extended active site region and anion-binding exosites (ABEs) I and II. For the current project, amide proton hydrogen-deuterium exchange coupled with MALDI-TOF mass spectrometry was used to characterize ligand binding to individual exosites and to investigate the presence of exosite-active site and exosite-exosite interactions. PAR3(44–56) and PAR1(49–62) were observed to bind to thrombin ABE I and then to exhibit long range effects over to ABE II. By contrast, Hirudin(54–65) focused more on ABE I and did not transmit influences over to ABE II. Although these three ligands were each directed to ABE I, they did not promote the same conformational consequences. d-Phe-Pro-Arg-chloromethyl ketone inhibition at the thrombin active site led to further local and long range consequences to thrombin-ABE I ligand complexes with the autolysis loop often most affected. When Hirudin(54–65) was bound to ABE I, it was still possible to bind GpIbα(269–286) or fibrinogen γ′(410–427) to ABE II. Each ligand exerted its predominant influences on thrombin and also allowed interexosite communication. The results obtained support the proposal that thrombin is a highly dynamic protein. The transmission of ligand-specific local and long range conformational events is proposed to help regulate this multifunctional enzyme.     

The challenge of the proteome dynamic range and its implications for in‐depth proteomics

The dynamic range of the cellular proteome approaches seven orders of magnitude—from one copy per cell to ten million copies per cell. Since a proteome's abundance distribution represents a nearly symmetric bell‐shape curve on the logarithmic copy number scale, detection of half of the expressed cellular proteome, i.e. approximately 5000 proteins, should be a relatively straightforward task with modern mass spectrometric instrumentation that exhibits four orders of magnitude of the dynamic range, while deeper proteome analysis should be progressively more difficult. Indeed, metaanalysis of 15 recent papers that claim detection of >5000 protein groups reveals that the half‐proteome analyses currently requires ≈5 h of chromatographic separation, while deeper analyses yield on average ≤20 new proteins per hour of chromatographic gradient. Therefore, a typical proteomics experiment consists of a “high‐content” part, with the detection rate of approximately 1000 proteins/h, and a “low‐content” tail with much lower rate of discovery and respectively, lower cost efficiency. This result calls for disruptive innovation in deep proteomics analysis.     

From proteins to systems: the British Society for Proteomics Research (BSPR) meeting 2005

This report describes the highlights of the second scientific meeting of the British Society for Proteome Research (BSPR), jointly organised with the European Bioinformatics Institute (EBI), and held at The Genome Centre, Cambridge UK in July 2005. The theme of the meeting was "From Proteins to Systems" covering many diverse aspects of proteomics, bioinformatics and systems biology.     

A Proteomic Strategy Identifies Lysine Methylation of Splicing Factor snRNP70 by the SETMAR Enzyme

The lysine methyltransferase (KMT) SETMAR is implicated in the response to and repair of DNA damage, but its molecular function is not clear. SETMAR has been associated with dimethylation of histone H3 lysine 36 (H3K36) at sites of DNA damage. However, SETMAR does not methylate H3K36 in vitro. This and the observation that SETMAR is not active on nucleosomes suggest that H3K36 methylation is not a physiologically relevant activity. To identify potential non-histone substrates, we utilized a strategy on the basis of quantitative proteomic analysis of methylated lysine. Our approach identified lysine 130 of the mRNA splicing factor snRNP70 as a SETMAR substrate in vitro, and we show that the enzyme primarily generates monomethylation at this position. Furthermore, we show that SETMAR methylates snRNP70 Lys-130 in cells. Because snRNP70 is a key early regulator of 5′ splice site selection, our results suggest a model in which methylation of snRNP70 by SETMAR regulates constitutive and/or alternative splicing. In addition, the proteomic strategy described here is broadly applicable and is a promising route for large-scale mapping of KMT substrates.     

Plasticity of cytochrome P450 2B4 as investigated by hydrogen-deuterium exchange mass spectrometry and X-ray crystallography

Crystal structures of the xenobiotic metabolizing cytochrome P450 2B4 have demonstrated markedly different conformations in the presence of imidazole inhibitors or in the absence of ligand. However, knowledge of the plasticity of the enzyme in solution has remained scant. Thus, hydrogen-deuterium exchange mass spectrometry (DXMS) was utilized to probe the conformations of ligand-free P450 2B4 and the complex with 4-(4-chlorophenyl)imidazole (4-CPI) or 1-biphenyl-4-methyl-1H-imidazole (1-PBI). The results of DXMS indicate that the binding of 4-CPI slowed the hydrogen-deuterium exchange rate over the B'- and C-helices and portions of the F-G-helix cassette compared with P450 2B4 in the absence of ligands. In contrast, there was little difference between the ligand-free and 1-PBI-bound exchange sets. In addition, DXMS suggests that the ligand-free P450 2B4 is predominantly open in solution. Interestingly, a new high resolution structure of ligand-free P450 2B4 was obtained in a closed conformation very similar to the 4-CPI complex. Molecular dynamics simulations performed with the closed ligand-free structure as the starting point were used to probe the energetically accessible conformations of P450 2B4. The simulations were found to equilibrate to a conformation resembling the 1-PBI-bound P450 2B4 crystal structure. The results indicate that conformational changes observed in available crystal structures of the promiscuous xenobiotic metabolizing cytochrome P450 2B4 are consistent with its solution structural behavior.     

Accumulation of newly synthesized F1 in vivo in arabidopsis mitochondria provides evidence for modular assembly of the plant F1Fo ATP synthase

F(1) subcomplex in mitochondrial samples is often considered to be a breakage product of the F(1)F(O) ATP synthase during sample handling and electrophoresis. We have used a progressive (15)N incorporation strategy to investigate the plant F(1)F(O) ATP synthase assembly model and the apparently free F(1) in plant mitochondria which is found in both the inner membrane and matrix. We show that subunits within F(1) in the inner membrane and matrix had a relatively higher (15)N incorporation rate than corresponding subunits in intact membrane F(1)F(O). This demonstrates that free F(1) was a newer pool with a faster turnover rate consistent with it being an assembly intermediate in vivo. Import of [(35)S]Met-labeled F(1) subunit precursors into Arabidopsis mitochondria showed the rapid accumulation of F(1) assembly intermediates. The different (15)N incorporation rate in matrix F(1), inner membrane F(1) and intact F(1)F(O) demonstrates these three represent different protein populations and are likely step by step intermediates during the assembly process of plant mitochondrial ATP synthase. The potential biological implications of in vivo accumulation of enzymatically active F(1) in mitochondria are discussed.     

Characterization of the Prion Protein in Human Urine

The presence of the prion protein (PrP) in normal human urine is controversial and currently inconclusive. This issue has taken a special relevance because prion infectivity has been demonstrated in urine of animals carrying experimental or naturally occurring prion diseases, but the actual presence and tissue origin of the infectious prion have not been determined. We used immunoprecipitation, one- and two-dimensional electrophoresis, and mass spectrometry to prove definitely the presence of PrP in human urine and its post-translational modifications. We show that urinary PrP (uPrP) is truncated mainly at residue 112 but also at other residues up to 122. This truncation makes uPrP undetectable with some commonly used antibodies to PrP. uPrP is glycosylated and carries an anchor which, at variance with that of cellular PrP, lacks the inositol-associated phospholipid moiety, indicating that uPrP is probably shed from the cell surface. The detailed characterization of uPrP reported here definitely proves the presence of PrP in human urine and will help determine the origin of prion infectivity in urine.     

Proteome-wide Analysis of Protein Thermal Stability in the Model Higher Plant Arabidopsis thaliana

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Highlights

• •Over 1700 Arabidopsis proteins with thermal models in multiple replicates.
• •Melting temperature correlates with 1°, 2°, and 3° protein characteristics.
• •Ligand-induced thermal shifts are evident in complex protein extracts.