Survey of peptide quantification methods and comparison of their reproducibility: A case study using oxytocin.

USP's peptide reference standards content is typically determined using an HPLC assay against an external standard for which the purity was determined by a mass balance approach. To explore the use of other analytical methods, the USP Biologics Department conducted a multi-laboratory collaborative study. The study determined the inter-laboratory variability for peptide quantitation using the following methods: HPLC assay, quantitative nuclear magnetic resonance (qNMR) spectroscopy, or amino acid analysis (AAA). The three methods were compared with regard to their suitability for quantitation of the nonapeptide oxytocin. In this study, the HPLC assay method using the same peptide bulk material as the standard showed the lowest inter-lab variability. The coefficient of variation (%CV) was calculated without counting the uncertainty associated with the purity assignment of the standard with mass balance. The proton qNMR method is a direct measurement of the peptide against an internal standard, which is not difficult to perform under common laboratory conditions. Because of the simpler operation and shorter analytical time, qNMR as a primary method for peptide reference standard value assignment deserves further exploration.

Overlap of proteome changes in Medicago truncatula in response to auxin and Sinorhizobium meliloti.

We used proteome analysis to identify proteins induced during nodule initiation and in response to auxin in Medicago truncatula. From previous experiments, which found a positive correlation between auxin levels and nodule numbers in the M. truncatula supernodulation mutant sunn (supernumerary nodules), we hypothesized (1) that auxin mediates protein changes during nodulation and (2) that auxin responses might differ between the wild type and the supernodulating sunn mutant during nodule initiation. Increased expression of the auxin response gene GH3:beta-glucuronidase was found during nodule initiation in M. truncatula, similar to treatment of roots with auxin. We then used difference gel electrophoresis and tandem mass spectrometry to compare proteomes of wild-type and sunn mutant roots after 24 h of treatment with Sinorhizobium meliloti, auxin, or a control. We identified 131 of 270 proteins responding to treatment with S. meliloti and/or auxin, and 39 of 89 proteins differentially displayed between the wild type and sunn. The majority of proteins changed similarly in response to auxin and S. meliloti after 24 h in both genotypes, supporting hypothesis 1. Proteins differentially accumulated between untreated wild-type and sunn roots also showed changes in auxin response, consistent with altered auxin levels in sunn. However, differences between the genotypes after S. meliloti inoculation were largely not due to differential auxin responses. The role of the identified candidate proteins in nodule initiation and the requirement for their induction by auxin could be tested in future functional studies.

Low temperature treatment at the young microspore stage induces protein changes in rice anthers.

Male reproductive development in rice is very sensitive to various forms of environmental stresses including low temperature. A few days of cold treatment (<20 degrees C) at the young microspore stage induce severe pollen sterility and thus large grain yield reductions. To investigate this phenomenon, anther proteins at the early stages of microspore development, with or without cold treatment at 12 degrees C, were extracted, separated by two-dimensional gel electrophoresis, and compared. The cold-sensitive cultivar Doongara and the relatively cold-tolerant cultivar HSC55 were used. The abundance of 37 anther proteins was changed more than 2-fold after 1, 2, and 4 days of cold treatment in cv. Doongara. Among them, one protein was newly induced, 32 protein spots were up-regulated, and four protein spots were down-regulated. Of these 37 protein spots, we identified two anther-specific proteins (putative lipid transfer protein and Osg6B) and a calreticulin that were down-regulated and a cystine synthase, a beta-6 subunit of the 20 S proteasome, an H protein of the glycine cleavage system, cytochrome c oxidase subunit VB, an osmotin protein homologue, a putative 6-phosphogluconolactonase, a putative adenylate kinase, a putative cysteine proteinase inhibitor, ribosomal protein S12E, a caffeoyl-CoA O-methyltransferase, and a monodehydroascorbate reductase that were up-regulated. Identification of these proteins is available upon request. Accumulation of these proteins did not vary greatly after cold treatment in panicles of cv. Doongara or in the anthers of the cv. HSC55. The newly induced protein named Oryza sativa cold-induced anther protein (OsCIA) was identified as an unknown protein. The OsCIA protein was detected in panicles, leaves, and seedling tissues under normal growth conditions. Quantitative real time RT-PCR analysis of OsCIA mRNA expression showed no significant change between low temperature-treated and untreated plants. A possible regulatory role for the newly induced protein is proposed.

Effect of early cold stress on the maturation of rice anthers.

Male reproductive development in rice (Oryza sativa Linnaeus is very sensitive to various forms of environmental stresses including low temperature. Here, we present our findings on the proteomic analysis of the later developmental consequences of low temperature treatment on rice anthers. Anther proteins at the trinucleate stage, with or without cold treatment for four days at 12 degrees C at the young microspore stage, were extracted, separated by two-dimensional gel electrophoresis (2-DE) and compared. More than 3000 rice anther proteins of cold-sensitive cultivar Doongara plants at the trinucleate stage were resolved on 2-DE gels over a pH range of 4-7 and detected by silver-staining. Seventy protein spots were differentially displayed after four days of cold treatment at the young microspore stage. Of these, 12 protein spots were newly-induced, 47 were up-regulated, and 11 were down-regulated by cold treatment at the early microspore stage. We identified 18 by matrix-assisted laser desorption/ionization mass spectrometry time of flight (MALDI-TOF) analysis. Of the identified proteins, seven were observed as breakdown (cleavage) products by a combination of 2-DE and MALDI-TOF analysis, thus demonstrating for the first time that cold temperature stress at the young microspore stage enhances and induces partial degradation of proteins in the rice anthers at the trinucleate stage.

Proteome analysis of male gametophyte development in rice anthers.

We used proteomic analysis to investigate the changing patterns of protein synthesis during pollen development in anthers from rice plants grown under strictly controlled growth conditions. Cytological analysis and external growth measurements such as anther length, auricle distances and days before flowering were used to determine pollen developmental stages. This allowed the collection of synchronous anther materials representing six discrete pollen developmental stages. Proteins were extracted from the anther samples and separated by two-dimensional gel electrophoresis to produce proteome maps. The anther proteome maps of different developmental stages were compared and 150 protein spots, which were changed consistently during development, were analysed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry to produce peptide mass fingerprint (PMF) data. Database searches using these PMF data revealed the identities of 40 of the protein spots analyzed. These 40 proteins represent 33 unique gene products. Four protein spots that could not be identified by PMF analysis were analysed by N-terminal microsequencing. Multiple charge-isoforms of vacuolar acid invertase, fructokinase, beta-expansin and profilin were identified. These proteins are closely associated with sugar metabolism, cell elongation and cell expansion, all of which are cell activities that are essential to pollen germination. The existence of multiple isoforms of the same proteins suggests that during the process of pollen development some kind of post-translational modification of these proteins occurs.

Proteomic analysis reveals developmentally expressed rice homologues of grass group II pollen allergens.

Three isoallergens of Ory s 2, homologues of grass group II pollen allergens, were identified from rice and characterised by proteome and immunochemical analyses. The N-terminal amino acid sequence profiles of three proteins on a 2-dimensional electrophoresis (2-DE) gel of rice pollen proteins matched 100% to the protein sequences encoded by three rice expressed sequence tags (ESTs). The deduced protein sequences from these ESTs share sequence identities of 41-43% with the protein sequences of the group II pollen allergens of different grasses, and sequence identity of 39% with the C-terminal portion of rice group I pollen allergens. Signal peptide sequences, which are similar to the leader peptides of other major pollen allergens, are also present in the deduced amino acid sequences. Polyclonal antibodies, produced in rabbits using Ory s 2 proteins purified by 2-DE, were used to investigate the developmental-stage- and tissue-specific expression of Ory s 2 by immunochemical analysis. Results of immunochemical experiments show that Ory s 2 proteins are expressed only at the late stage of pollen development and they do not have cross-reactivity with group II pollen allergens from some other common grasses.

Evaluation of proteome reference maps for cross-species identification of proteins by peptide mass fingerprinting.

We tested whether proteome reference maps established for one species can be used for cross-species protein identification by comparing two-dimensional protein gel patterns and protein identification data of two closely related bacterial strains and four plant species. First, proteome profiles of two strains of the fully sequenced bacterium Sinorhizobium meliloti were compared as an example of close relatedness, high reproducibility and sequence availability. Secondly, the proteome profiles of three legumes (Medicago truncatula, Melilotus alba and Trifolium subterraneum), and the nonlegume rice (Oryza sativa) were analysed to test cross-species similarities. In general, we found stronger similarities in gel patterns of the arrayed proteins between the two bacterial strains and between the plant species than could be expected from the sequence similarities. However, protein identity could not be concluded from their gel position, not even when comparing strains of the same species. Surprisingly, in the bacterial strains peptide mass fingerprinting was more reliable for species-specific protein identification than N-terminal sequencing. While peptide masses were found to be unreliable for cross-species protein identification, we present useful criteria to determine confident matching against species-specific expressed sequence tag databases. In conclusion, we present evidence that cautions the use of proteome reference maps and peptide mass fingerprinting for cross-species protein identification.