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Author(s): Jane L. Hurst (corresponding author) [1]; Caroline E. Payne [1]; Charlotte M. Nevison [1]; Amr D. Marie [2]; Richard E. Humphries [1]; Duncan H. L. Robertson [2]; Andrea Cavaggioni [...]

Many rodents are now known to exhibit an obligate proteinuria that delivers urine-mediated chemosignals. In this paper, we explore the urinary proteins of the Roborovski hamster (Phodopus roborovskii). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of urine from individual male and female Roborovski hamsters revealed 2 proteins, with approximate masses of 6 and 17 kDa, the expression pattern of which showed little variation between individuals or between sexes. Peptide mass fingerprints obtained from these 2 proteins revealed a number of features: 1) the proteins of a given mass were the same in all individuals regardless of sex, 2) the 6 kDa protein was not a fragment of the 21 kDa protein, and 3) neither protein was a fragment of a larger, conserved protein such as serum albumin. Electrospray mass spectrometry of purified protein preparations established the mass of the larger protein as invariant, at 17144 ± 2 Da in all samples. This protein has been termed roborovskin. The primary structure of roborovskin was determined by tandem mass spectrometry of peptides derived from independent and overlapping digestion with 3 proteases, supported by Edman degradation of the protein N-terminus. Roborovskin shared significant homology with olfactory-binding proteins from Myodes glareolus (bank vole) and with aphrodisin and submandibular protein from the golden hamster Mesocricetus auratus, all of which belong to the lipocalin superfamily. Lower levels of homology were also indicated between a variety of other lipocalins including the major urinary proteins from house mice and Norway rats. A model of the tertiary structure of roborovskin was constructed from the primary sequence by homology modeling. This model structure resembled other 8-stranded beta barrel lipocalins. Thus, the Roborovski hamster may demonstrate another variant of urinary lipocalin expression, as for the animals studied here, there appears to be no polymorphism in expression either between sexes or individuals.

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.

Analysis of intact protein mixtures by electrospray ionization mass spectrometry requires the resolution of a complex, overlapping set of multiply charged envelopes. To ascertain the ability of a moderate resolution mass spectrometer to resolve such mixtures, we have analyzed the soluble proteins of adult chick skeletal muscle. This is a highly specialized tissue showing a marked bias in expression of glycolytic enzymes in the soluble fraction. SDS-PAGE-resolved proteins were first identified by a combination of matrix-assisted laser desorption ionization time-of-flight (TOF) and electrospray ionization tandem mass spectrometry. Then the mixture of intact proteins was introduced into the electrospray source of a Q-TOF mass spectrometer either by direct infusion or via a C4 desalting trap. In both instances, the complex pattern of peaks could be resolved into true masses, and these masses could in many instances be reconciled with the masses predicted from the known protein sequences when qualified by expected co- and post-translational modifications. These included loss of the N-terminal initiator methionine residue and N-terminal acetylation. The ability to resolve such a complex mixture of proteins with a routine instrument is of considerable value in analyses of protein expression and in the confirmation of post-translational changes in mature proteins.

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.