Duncan H. L. Robertson

Dynamics of Protein Turnover, a Missing Dimension in Proteomics

Functional genomic experiments frequently involve a comparison of the levels of gene expression between two or more genetic, developmental, or physiological states. Such comparisons can be carried out at either the RNA (transcriptome) or protein (proteome) level, but there is often a lack of congruence between parallel analyses using these two approaches. To fully interpret protein abundance data from proteomic experiments, it is necessary to understand the contributions made by the opposing processes of synthesis and degradation to the transition between the states compared. Thus, there is a need for reliable methods to determine the rates of turnover of individual proteins at amounts comparable to those obtained in proteomic experiments. Here, we show that stable isotope-labeled amino acids can be used to define the rate of breakdown of individual proteins by inspection of mass shifts in tryptic fragments. The approach has been applied to an analysis of abundant proteins in glucose-limited yeast cells grown in aerobic chemostat culture at steady state. The average rate of degradation of 50 proteins was 2.2%/h, although some proteins were turned over at imperceptible rates, and others had degradation rates of almost 10%/h. This range of values suggests that protein turnover is a significant missing dimension in proteomic experiments and needs to be considered when assessing protein abundance data and comparing it to the relative abundance of cognate mRNA species.

Positional proteomics: selective recovery and analysis of N-terminal proteolytic peptides.

Bottom-up proteomics is the analysis of peptides derived from single proteins or protein mixtures, and because each protein generates tens of peptides, there is scope for controlled reduction in complexity. We report here a new strategy for selective isolation of the N-terminal peptides of a protein mixture, yielding positionally defined peptides. The method is tolerant of several fragmentation methods, and the databases that must be searched are substantially less complex.

Absolute Multiplexed Quantitative Analysis of Protein Expression during Muscle Development Using QconCAT

Stable isotope-labeled proteotypic peptides are used as surrogate standards for absolute quantification of proteins in proteomics. However, a stable isotope-labeled peptide has to be synthesized, at relatively high cost, for each protein to be quantified. To multiplex protein quantification, we developed a method in which gene design de novo is used to create and express artificial proteins (QconCATs) comprising a concatenation of proteotypic peptides. This permits absolute quantification of multiple proteins in a single experiment. This complete study was constructed to define the nature, sources of error, and statistical behavior of a QconCAT analysis. The QconCAT protein was designed to contain one tryptic peptide from 20 proteins present in the soluble fraction of chicken skeletal muscle. Optimized DNA sequences encoding these peptides were concatenated and inserted into a vector for high level expression in Escherichia coli. The protein was expressed in a minimal medium containing amino acids selectively labeled with stable isotopes, creating an equimolar series of uniformly labeled proteotypic peptides. The labeled QconCAT protein, purified by affinity chromatography and quantified, was added to a homogenized muscle preparation in a known amount prior to proteolytic digestion with trypsin. As anticipated, the QconCAT was completely digested at a rate far higher than the analyte proteins, confirming the applicability of such artificial proteins for multiplexed quantification. The nature of the technical variance was assessed and compared with the biological variance in a complete study. Alternative ionization and mass spectrometric approaches were investigated, particularly LC-ESI-TOF MS and MALDI-TOF MS, for analysis of proteins and tryptic peptides. QconCATs offer a new and efficient approach to precise and simultaneous absolute quantification of multiple proteins, subproteomes, or even entire proteomes.

Unravelling the chemical basis of competitive scent marking in house mice

Major urinary proteins (MUPs) in the urine of male house mice, Mus domesticus, bind the male signalling volatiles 2- sec -butyl-4,5-dihydrothiazole (thiazole) and 3,4-dehydro- exo -brevicomin (brevicomin) and slowly release these volatiles from urinary scent marks. To examine the role of urinary proteins and volatiles, either attached or unattached to the proteins, in competitive scent marking, we fractionated urine from isolated male BALB/c laboratory mice, Mus musculus, by size-exclusion chromatography into three pools. Pool I contained all of the urinary proteins and their bound ligands while pools II and III contained lower molecular weight components including unbound signalling volatiles. In experiment 1, pools I-III were streaked out on to absorbent paper (Benchkote) and introduced into enclosures housing single wild-caught male mice, together with a clean control surface. Each male was tested with fresh stimuli and with aged stimuli deposited 24 h previously. Only pool I stimulated significantly more countermarking and investigation than the control, attracting mice to investigate from a distance even when the rate of ligand release was considerably reduced after 24 h. Experiment 2 examined responses to pool I when this was fresh, aged by 7 days, or had been mixed with menadione to displace ligands from the proteins. Although all three protein stimuli were investigated and countermarked more than a clean control, the aged and menadione-treated pool I stimulated the strongest responses, despite containing the lowest levels of thiazole and brevicomin. Thus competitive countermarking is stimulated by proteins or by nonvolatile protein-ligand complexes in male urine, while release of volatile ligands attracts attention to a competitors scent marks. Copyright 1999 The Association for the Study of Animal Behaviour.

Extraction, characterization, and binding analysis of two pheromonally active ligands associated with major urinary protein of house mouse (Mus musculus).

Mouse urine contains substantial quantities of a family of proteins (MUPs) that are members of the lipocalycin family of proteins and that are potentially capable of binding hydrophobic molecules. We have used gas chromatography-mass spectrometry (GC-MS) to characterize two ligands associated with the MUPs, a thiazole and a brevicomin derivative. Previous work has suggested a role for these two ligands as androgen-dependent pheromones. In urine, nearly all of these ligands are protein bound and fractionation of MUPs on Mono-Q anion exchange chromatography indicated some specificity of ligand binding by the MUP subclasses.

Structural and functional differences in isoforms of mouse major urinary proteins: a male-specific protein that preferentially binds a male pheromone

The MUPs (major urinary proteins) of the house mouse, Mus domesticus, are lipocalins that bind and slowly release male-specific pheromones in deposited scent marks. However, females also express these proteins, consistent with a second role in encoding individual signatures in scent marks. We have purified and characterized an atypical MUP from the urine of male C57BL/6J inbred mice, which is responsible for the binding of most of the male pheromone, 2-sec-butyl-4,5-dihydrothiazole, and which is also responsible for the slow release of this pheromone from scent marks. This protein is absent from the urine of female mice of the same strain. The protein has been characterized by MS, leading to unequivocal identification as a previously uncharacterized gene product, providing compelling evidence for the expression of this gene in liver and manifestation in urine. These properties contrast strongly with those of the other MUPs in the same urine sample, and suggest that the requirement to manifest a male-specific pheromone has been met by evolution of a cognate protein specifically adapted to the binding and release of this ligand. This atypical MUP is also present in a random sample of wild-caught male mice, confirming that this protein is not specific to the inbred mouse strain but is present in natural populations also.

Polymorphism in major urinary proteins: molecular heterogeneity in a wild mouse population.

Major urinary proteins (MUPs) are present in high levels in the urine of mice, and the specific profile of MUPs varies considerably among wild-caught individuals. We have conducted a detailed study of the polymorphic variation within a geographically constrained island population, analyzing the MUP heterogeneity by isoelectric focusing and analytical ion exchange chromatography. Several MUPs were purified in sufficient quantities for analysis by electrospray ionization mass spectrometry and MALDI-TOF mass spectrometry of endopeptidase Lys-C peptide maps. The results of such analyses permitted the identification of three new MUP allelic variants. In each of these proteins, the sites of variation were located to a restricted segment of the polypeptide chain, projecting to a patch on the surface of the protein, and connected to the central lipocalin calyx through the polypeptide backbone. The restriction of the polymorphic variation to one segment of the polypeptide may be of functional significance, either in the modulation of ligand release or in communication of individuality signals within urinary scent marks.

Molecular Heterogeneity of Urinary Proteins in Wild House Mouse Populations

Major urinary proteins (MUPs) from the urine of individual wild mice were characterized using electrospray ionization mass spectrometry (ESI-MS) and compared to MUPs from the urine of inbred mice. The wild mice showed considerable variation between individuals in the expression of a group of MUPs with similar masses. Some individuals excreted MUPs of unique molecular mass whilst some failed to express MUPs seen commonly in the other individuals. All the wild individuals contained proteins not previously observed in inbred mice. Urine from one individual was fractionated using anion exchange chromatography prior to analysis by ESI-MS. By analysing urine from inbred samples under the same conditions it was possible to relate, using mass and net charge in solution, MUPs from the wild sample to the MUPs that have been observed previously in inbred strains. This has allowed tentative identification of some MUPs from the wild mouse. The effect of collection history of urine from wild mice was also investigated. ESI-MS analysis of MUPs in a faecally contaminated sample showed the loss of a C-terminal tripeptide when compared to an uncontaminated sample from the same mouse, consistent with the presence of a specific endopeptidase. Similarly a sample of pooled urine provided by twelve individuals trapped from the same population showed evidence of loss of the C-terminal dipeptide.

Effect of polymorphisms on ligand binding by mouse major urinary proteins

Mouse urine contains an abundance of major urinary proteins, lipocalins, whose roles include slow release of semiochemicals. These proteins are highly polymorphic, with small sequence differences between individual members. In this study, we purified to homogeneity four of these proteins from two strains of inbred mice and characterized them by mass spectrometry. This analysis has led to the discovery of another variant in this group of proteins. Three of the polymorphic variants that map to the surface have no effect on the binding of a fluorescent probe in the binding cavity, but the fourth, characterized by a Phe to Val substitution in the cavity, shows a substantially lower affinity and fluorescence yield for the probe. These results are interpreted in light of the known crystal structure of the protein and molecular modeling calculations, which rationalize the experimental findings. This work raises the possibility that the calyx‐binding site can show specificity for different ligands, the implications of which on pheromone binding and chemical communication are discussed.

Stable isotope labelling in vivo as an aid to protein identification in peptide mass fingerprinting.

Peptide mass fingerprinting (PMF) is a powerful technique for identification of proteins derived from in‐gel digests by virtue of their matrix‐assisted laser desorption/ionization‐time of flight mass spectra. However, there are circumstances where the under‐representation of peptides in the mass spectrum and the complexity of the source proteome mean that PMF is inadequate as an identification tool. In this paper, we show that identification is substantially enhanced by inclusion of composition data for a single amino acid. Labelling in vivo with a stable isotope labelled amino acid (in this paper, decadeuterated leucine) identifies the number of such amino acids in each digest fragment, and show a considerable gain in the ability of PMF to identify the parent protein. The method is tolerant to the extent of labelling, and as such, may be applicable to a range of single cell systems.