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Abstract Background Stagonospora nodorum, a fungal ascomycete in the class dothideomycetes, is a damaging pathogen of wheat. It is a model for necrotrophic fungi that cause necrotic symptoms via the interaction of multiple effector proteins with cultivar-specific receptors. A draft genome sequence and annotation was published in 2007. A second-pass gene prediction using a training set of 795 fully EST-supported genes predicted a total of 10762 version 2 nuclear-encoded genes, with an additional 5354 less reliable version 1 genes also retained. Results In this study, we subjected soluble mycelial proteins to proteolysis followed by 2D LC MALDI-MS/MS. Comparison of the detected peptides with the gene models validated 2134 genes. 62% of these genes (1324) were not supported by prior EST evidence. Of the 2134 validated genes, all but 188 were version 2 annotations. Statistical analysis of the validated gene models revealed a preponderance of cytoplasmic and nuclear localised proteins, and proteins with intracellular-associated GO terms. These statistical associations are consistent with the source of the peptides used in the study. Comparison with a 6-frame translation of the S. nodorum genome assembly confirmed 905 existing gene annotations (including 119 not previously confirmed) and provided evidence supporting 144 genes with coding exon frameshift modifications, 604 genes with extensions of coding exons into annotated introns or untranslated regions (UTRs), 3 new gene annotations which were supported by tblastn to NR, and 44 potential new genes residing within un-assembled regions of the genome. Conclusion We conclude that 2D LC MALDI-MS/MS is a powerful, rapid and economical tool to aid in the annotation of fungal genomic assemblies.

Immunoaffinity mass spectrometry as an approach for diagnostic biomarker assays combines the advantages of antibody selectivity with the multiplexing and analytical performance of mass spectrometry. A method has been developed to detect and quantify three protein biomarkers for a diabetic kidney disease prognostic assay, PromarkerD. The methodology reflects an immunoaffinity approach compatible with higher throughput and robust clinical application. After preparation and purification of antibody-bead conjugates for the three target proteins, an immunoaffinity capture step provides a solution for reduction, alkylation, and digestion on-bead. Targeted mass spectrometry provides a quantitative measure of each biomarker in a rapid 8 min run using a microflow LCMS workflow., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Aberrant protein glycosylation is a characteristic of diverse diseases which has been explored as biomarkers. To support translational serum glycoprotein biomarker discovery and validation, we developed a semi-automated workflow using individual lectin-coupled magnetic beads to conduct lectin pulldowns in a high-throughput format. Lectins are naturally occurring glycoprotein binding proteins widely used in glycobiology. While lectin-affinity isolation has been coupled to mass spectrometry-based proteomics, the lectin magnetic bead array (LeMBA) platform allows technically robust screening and measurement of clinical cohorts. This chapter describes detailed lectin-magnetic bead coupling, serum denaturation, lectin magnetic bead pulldown, and on-bead trypsin digest. The resulting tryptic peptides are analyzed by untargeted or targeted liquid chromatography-mass spectrometry (LC-MS), for biomarker discovery, or qualification/validation, respectively. LeMBA-MS generates quantitative data for glycoforms based on lectin affinity of the glycoprotein coupled with MS measurement of one or more prototypic peptides and has successfully been used to discover and validate novel serum cancer glycoprotein biomarkers. This chapter includes detailed protocols for two different liquid handlers, along with recommendations on quality control measures for clinical biomarker studies., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Phytophthora cinnamomi is a pathogenic oomycete that causes plant dieback disease across a range of natural ecosystems and in many agriculturally important crops on a global scale. An annotated draft genome sequence is publicly available (JGI Mycocosm) and suggests 26,131 gene models. In this study, soluble mycelial, extracellular (secretome), and zoospore proteins of P. cinnamomi were exploited to refine the genome by correcting gene annotations and discovering novel genes. By implementing the diverse set of sub-proteomes into a generated proteogenomics pipeline, we were able to improve the P. cinnamomi genome annotation. Liquid chromatography mass spectrometry was used to obtain high confidence peptides with spectral matching to both the annotated genome and a generated 6-frame translation. Two thousand seven hundred sixty-four annotations from the draft genome were confirmed by spectral matching. Using a proteogenomic pipeline, mass spectra were used to edit the P. cinnamomi genome and allowed identification of 23 new gene models and 60 edited gene features using high confidence peptides obtained by mass spectrometry, suggesting a rate of incorrect annotations of 3% of the detectable proteome. The novel features were further validated by total peptide support, alongside functional analysis including the use of Gene Ontology and functional domain identification. We demonstrated the use of spectral data in combination with our proteogenomics pipeline can be used to improve the genome annotation of important plant diseases and identify missed genes. This study presents the first use of spectral data to edit and manually annotate an oomycete pathogen., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Andronis, Hane, Bringans, Hardy, Jacques, Lipscombe and Tan.)

PromarkerD is a proteomics derived test for predicting diabetic kidney disease that measures the concentrations of three plasma protein biomarkers, APOA4, CD5L and IBP3. Antibodies against these proteins were developed and applied to a multiplexed immunoaffinity capture mass spectrometry assay. In parallel, and facilitating current clinical laboratory workflows, a standard ELISA was also developed to measure each protein. The performance characteristics of the two technology platforms were compared using a cohort of 100 samples, with PromarkerD test scores demonstrating a high correlation (R = 0.97). These technologies illustrate the potential for large scale, high throughput clinical applications of proteomics now and into the future.

Background: PromarkerD is a novel proteomics derived blood test for predicting diabetic kidney disease (DKD). The test is based on an algorithm that combines the measurement of three plasma protein biomarkers (CD5L, APOA4, and IBP3) with three clinical variables (age, HDL-cholesterol, and eGFR). The initial format of the assay used immunodepletion of plasma samples followed by targeted mass spectrometry (MRM-LCMS). The aim of this study was to convert the existing assay into an immunoaffinity approach compatible with higher throughput and robust clinical application., Methods: A newly optimised immunoaffinity-based assay was developed in a 96 well format with MRM measurements made using a low-flow LCMS method. The stability, reproducibility and precision of the assay was evaluated. A direct comparison between the immunoaffinity method and the original immunodepletion method was conducted on a 100-person cohort. Subsequently, an inter-lab study was performed of the optimised immunoaffinity method in two independent laboratories., Results: Processing of plasma samples was greatly simplified by switching to an immunoaffinity bead capture method, coupled to a faster and more robust microflow LCMS system. Processing time was reduced from seven to two days and the chromatography reduced from 90 to 8 min. Biomarker stability by temperature and time difference treatments passed acceptance criteria. Intra/Inter-day test reproducibility and precision were within 11% CV for all biomarkers. PromarkerD test results from the new immunoaffinity method demonstrated excellent correlation (R = 0.96) to the original immunodepletion method. The immunoaffinity assay was successfully transferred to a second laboratory (R = 0.98) demonstrating the robustness of the methodology and ease of method transfer., Conclusions: An immunoaffinity capture targeted mass spectrometry assay was developed and optimised. It showed statistically comparable results to those obtained from the original immunodepletion method and was also able to provide comparable results when deployed to an independent laboratory. Taking a research grade assay and optimising to a clinical grade workflow provides insights into the future of multiplex biomarker measurement with an immunoaffinity mass spectrometry foundation. In the current format the PromarkerD immunoaffinity assay has the potential to make a significant impact on prediction of diabetic kidney disease with consequent benefit to patients., Competing Interests: Competing interestsSB, JI, TC, ST, KP, RL are employees of Proteomics International, with SB and RL holding shares in the company. SP, OC and HE are employees of Atturos. Proteomics International is a beneficiary of patent PCT/AU2011/001212 that relates to biomarkers described in this manuscript., (© The Author(s) 2020.)

We report the results of a semi-quantitative peptide analysis of decomposition fluid under field-based conditions in the absence of a soil matrix. Sixteen domestic pig (Sus scrofa domesticus) cadavers were used to model human decomposition in trials conducted in the summer and winter months in Western Australia. Physical characteristics were recorded and targeted peptide components of decomposition fluid were analysed using high performance liquid chromatography-triple quadrupole mass spectrometry. Principal component analysis identified 29 peptides, originating from haemoglobin subunits alpha and beta, creatine kinase, beta-enolase and lactate dehydrogenase, that contributed to differences in the mean peak areas of samples collected during the early period of decomposition (days 6-12 and day 2 in winter and summer, respectively) and during the later period (days 24-34 and days 8-10 in winter and summer, respectively). Fold changes for 8 peptides between these periods were significantly different. Three peptides derived from haemoglobin subunit beta, one from beta-enolase and two from lactate dehydrogenase displayed consistent trends, in that a notable increase in mean peak area was followed by a marked decrease in both the summer and winter samples. When temperature was accounted for, these trends occurred at different time points in summer and winter, indicating that factors other than temperature had impacted the rate of degradation of the proteins involved. The single peptides derived from haemoglobin subunit alpha and creatine kinase displayed consistent increases in mean peak area for the summer samples, suggesting that temperature played the most significant role in their degradation. Further analyses revealed that 7 peptides (one originating from haemoglobin subunit alpha, three from haemoglobin subunit beta and three from lactate dehydrogenase) displayed consistent trends that could be correlated with total body score and with the early stages of decomposition. The consistent trends (mean peak area versus time) for peptides derived from several proteins during decomposition trials conducted under different temperature regimes further emphasised the potential of peptide analysis in time since death estimation., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Background and Aims: Resistance to the synthetic auxin 2,4-dichlorophenoxyacetic acid (2,4-D) in wild radish (Raphanus raphanistrum) appears to be due to a complex, multifaceted mechanism possibly involving enhanced constitutive plant defence and alterations in auxin signalling. Based on a previous gene expression analysis highlighting the plasma membrane as being important for 2,4-D resistance, this study aimed to identify the components of the leaf plasma membrane proteome that contribute to resistance., Methods: Isobaric tagging of peptides was used to compare the plasma membrane proteomes of a 2,4-D-susceptible and a 2,4-D-resistant wild radish population under control and 2,4-D-treated conditions. Eight differentially abundant proteins were then targeted for quantification in the plasma membranes of 13 wild radish populations (two susceptible, 11 resistant) using multiple reaction monitoring., Key Results: Two receptor-like kinases of unknown function (L-type lectin domain-containing receptor kinase IV.1-like and At1g51820-like) and the ATP-binding cassette transporter ABCB19, an auxin efflux transporter, were identified as being associated with auxinic herbicide resistance. The variability between wild radish populations suggests that the relative contributions of these candidates are different in the different populations., Conclusions: To date, no receptor-like kinases have been reported to play a role in 2,4-D resistance. The lectin-domain-containing kinase may be involved in perception of 2,4-D at the plasma membrane, but its ability to bind 2,4-D and the identity of its signalling partner(s) need to be confirmed experimentally. ABCB19 is known to export auxinic compounds, but its role in 2,4-D resistance in wild radish appears to be relatively minor., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

We report the peptide content of decomposition fluid produced under field-based conditions and in the absence of a soil matrix. Sixteen domestic pig (Sus scrofa domesticus) cadavers were used to model human decomposition in trials conducted in the summer and winter months in Western Australia. Physical characteristics were recorded and the peptide components of decomposition fluid were analysed using high performance liquid chromatography-time of flight mass spectrometry. A range of peptides was consistently detected in both summer and winter. Thirty seven peptides were common to both trials; 22 originating from haemoglobin subunit beta, 1 from haemoglobin subunit alpha, 4 from beta-enolase, and 2 from creatine kinase. In agreement with our previous findings, 13 peptides occurred consistently, regardless of trial conditions. Degradation patterns for haemoglobin subunits alpha and beta in summer and winter were similar when expressed in ADD and when adjusted for differences in temperature. The consistent identification of several protein-specific peptides generated during decomposition trials conducted under different temperature and rainfall regimes suggests that quantitative peptide analysis may be useful in estimating time since death., (Copyright © 2019 Elsevier B.V. All rights reserved.)

We report the preliminary observations of the peptide content of decomposition fluid produced under controlled laboratory conditions and in the absence of a soil matrix. Four domestic pig (Sus scrofa domesticus) cadavers were used to model human decomposition over a four-week trial period; physical characteristics were recorded and the peptide components of decomposition fluid was analysed using high performance liquid chromatography-time of flight mass spectrometry. Preliminary data analysis indicated that a range of peptides were consistently detected across the course of the trial period and 27 of these were common to all four cadavers; 22 originating from haemoglobin. The peptides associated with haemoglobin subunit alpha and beta displayed a breakdown pattern that remained consistent for all cadavers for the duration of the trial. Though identification of peptides during decomposition has potential for estimating the time since death, quantification of selected peptides is likely to be essential to identify time-dependent trends., (Copyright © 2019 Elsevier B.V. All rights reserved.)

A simple, selective and accurate reverse phase HPLC method was developed for detection and quantitation of γ-conglutin from lupin seed extract. A linear gradient of water and acetonitrile containing trifluoroacetic acid (TFA) on a reverse phase column (Agilent Zorbax 300SB C-18), with a flow rate of 0.8ml/min was able to produce a sharp and symmetric peak of γ-conglutin with a retention time at 29.16min. The identity of γ-conglutin in the peak was confirmed by mass spectrometry (MS/MS identification) and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) analysis. The data obtained from MS/MS analysis was matched against the specified database to obtain the exact match for the protein of interest. The proposed method was validated in terms of specificity, linearity, sensitivity, precision, recovery and accuracy. The analytical parameters revealed that the validated method was capable of selectively performing a good chromatographic separation of γ-conglutin from the lupin seed extract with no interference of the matrix. The detection and quantitation limit of γ-conglutin were found to be 2.68μg/ml and 8.12μg/ml respectively. The accuracy (precision and recovery) analysis of the method was conducted under repeatable conditions on different days. Intra-day and inter-day precision values less than 0.5% and recovery greater than 97% indicated high precision and accuracy of the method for analysis of γ-conglutin. The method validation findings were reproducible and can be successfully applied for routine analysis of γ-conglutin from lupin seed extract., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Lupins are important grain legume crops that form a critical part of sustainable farming systems, reducing fertilizer use and providing disease breaks. It has a basal phylogenetic position relative to other crop and model legumes and a high speciation rate. Narrow-leafed lupin (NLL; Lupinus angustifolius L.) is gaining popularity as a health food, which is high in protein and dietary fibre but low in starch and gluten-free. We report the draft genome assembly (609 Mb) of NLL cultivar Tanjil, which has captured >98% of the gene content, sequences of additional lines and a dense genetic map. Lupins are unique among legumes and differ from most other land plants in that they do not form mycorrhizal associations. Remarkably, we find that NLL has lost all mycorrhiza-specific genes, but has retained genes commonly required for mycorrhization and nodulation. In addition, the genome also provided candidate genes for key disease resistance and domestication traits. We also find evidence of a whole-genome triplication at around 25 million years ago in the genistoid lineage leading to Lupinus. Our results will support detailed studies of legume evolution and accelerate lupin breeding programmes., (© 2016 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

The abundant proteins in human milk have been well characterized and are known to provide nutritional, protective, and developmental advantages to both term and preterm infants. However, relatively little is known about the expression of the low abundance proteins that are present in human milk because of the technical difficulties associated with their detection. We used a combination of electrophoretic techniques, ProteoMiner treatment, and two-dimensional liquid chromatography to examine the proteome of human skim milk expressed between 7 and 28 days postpartum by healthy term mothers and identified 415 in a pooled milk sample. Of these, 261 were found in human skim milk for the first time, greatly expanding our understanding of the human skim milk proteome. The majority of the proteins identified were involved in either the immune response (24%) or in cellular (28%) or protein (16%) metabolism. We also used iTRAQ analysis to examine the effects of premature delivery on milk protein composition. Differences in protein expression between pooled milk from mothers delivering at term (38-41 weeks gestation) and preterm (28-32 weeks gestation) were investigated, with 55 proteins found to be differentially expressed with at least 90% confidence. Twenty-eight proteins were present at higher levels in preterm milk, and 27 were present at higher levels in term milk.

Mutations in the skeletal muscle α-actin gene (ACTA1) cause a range of pathologically defined congenital myopathies. Most patients have dominant mutations and experience severe skeletal muscle weakness, dying within one year of birth. To determine mutant ACTA1 pathobiology, transgenic mice expressing ACTA1(D286G) were created. These Tg(ACTA1)(D286G) mice were less active than wild-type individuals. Their skeletal muscles were significantly weaker by in vitro analyses and showed various pathological lesions reminiscent of human patients, however they had a normal lifespan. Mass spectrometry revealed skeletal muscles from Tg(ACTA1)(D286G) mice contained ∼25% ACTA1(D286G) protein. Tg(ACTA1)(D286G) mice were crossed with hemizygous Acta1(+/-) knock-out mice to generate Tg(ACTA1)(D286G)(+/+).Acta1(+/-) offspring that were homozygous for the transgene and hemizygous for the endogenous skeletal muscle α-actin gene. Akin to most human patients, skeletal muscles from these offspring contained approximately equal proportions of ACTA1(D286G) and wild-type actin. Strikingly, the majority of these mice presented with severe immobility between postnatal Days 8 and 17, requiring euthanasia. Their skeletal muscles contained extensive structural abnormalities as identified in severely affected human patients, including nemaline bodies, actin accumulations and widespread sarcomeric disarray. Therefore we have created valuable mouse models, one of mild dominant ACTA1 disease [Tg(ACTA1)(D286G)], and the other of severe disease, with a dramatically shortened lifespan [Tg(ACTA1)(D286G)(+/+).Acta1(+/-)]. The correlation between mutant ACTA1 protein load and disease severity parallels effects in ACTA1 families and suggests altering this ratio in patient muscle may be a therapy for patients with dominant ACTA1 disease. Furthermore, ringbinden fibres were observed in these mouse models. The presence of such features suggests that perhaps patients with ringbinden of unknown genetic origin should be considered for ACTA1 mutation screening. This is the first experimental, as opposed to observational, evidence that mutant protein load determines the severity of ACTA1 disease.

The G protein alpha-subunit (Gna1) in the wheat pathogen Stagonospora nodorum has previously been shown to be a critical controlling element in disease ontogeny. In this study, iTRAQ and 2-D LC MALDI-MS/MS have been used to characterise protein expression changes in the S. nodorum gna1 strain versus the SN15 wild-type. A total of 1336 proteins were identified. The abundance of 49 proteins was significantly altered in the gna1 strain compared with the wild-type. Gna1 was identified as having a significant regulatory role on primary metabolic pathways, particularly those concerned with NADPH synthesis or consumption. Mannitol dehydrogenase was up-regulated in the gna1 strain while mannitol 1-phosphate dehydrogenase was down-regulated providing direct evidence of Gna1 regulation over this enigmatic pathway. Enzymatic analysis and growth assays confirmed this regulatory role. Several novel hypothetical proteins previously associated with stress and pathogen responses were identified as positively regulated by Gna1. A short-chain dehydrogenase (Sch3) was also significantly less abundant in the gna1 strains. Sch3 was further characterised by gene disruption in S. nodorum by homologous recombination. Functional characterisation of the sch3 strains revealed their inability to sporulate in planta providing a further link to Gna1 signalling and asexual reproduction. These data add significantly to the identification of the regulatory targets of Gna1 signalling in S. nodorum and have demonstrated the utility of iTRAQ in dissecting signal transduction pathways.

Background: Stagonospora nodorum, a fungal ascomycete in the class dothideomycetes, is a damaging pathogen of wheat. It is a model for necrotrophic fungi that cause necrotic symptoms via the interaction of multiple effector proteins with cultivar-specific receptors. A draft genome sequence and annotation was published in 2007. A second-pass gene prediction using a training set of 795 fully EST-supported genes predicted a total of 10762 version 2 nuclear-encoded genes, with an additional 5354 less reliable version 1 genes also retained., Results: In this study, we subjected soluble mycelial proteins to proteolysis followed by 2D LC MALDI-MS/MS. Comparison of the detected peptides with the gene models validated 2134 genes. 62% of these genes (1324) were not supported by prior EST evidence. Of the 2134 validated genes, all but 188 were version 2 annotations. Statistical analysis of the validated gene models revealed a preponderance of cytoplasmic and nuclear localised proteins, and proteins with intracellular-associated GO terms. These statistical associations are consistent with the source of the peptides used in the study. Comparison with a 6-frame translation of the S. nodorum genome assembly confirmed 905 existing gene annotations (including 119 not previously confirmed) and provided evidence supporting 144 genes with coding exon frameshift modifications, 604 genes with extensions of coding exons into annotated introns or untranslated regions (UTRs), 3 new gene annotations which were supported by tblastn to NR, and 44 potential new genes residing within un-assembled regions of the genome., Conclusion: We conclude that 2D LC MALDI-MS/MS is a powerful, rapid and economical tool to aid in the annotation of fungal genomic assemblies.

As with many skeletal muscle diseases, the extraocular muscles (EOMs) are spared in skeletal muscle alpha-actin diseases, with no ophthalmoplegia even in severely affected patients. We hypothesised that the extraocular muscles sparing in these patients was due to significant expression of cardiac alpha-actin, the alpha-actin isoform expressed in heart and foetal skeletal muscle. We have shown by immunochemistry, Western blotting and a novel MRM-mass spectrometry technique, comparable levels of cardiac alpha-actin in the extraocular muscles of human, pig and sheep to those in the heart. The sparing of extraocular muscles in skeletal muscle alpha-actin disease is thus probably due to greater levels of cardiac alpha-actin, than the negligible amounts in skeletal muscles, diluting out the effects of the mutant skeletal muscle alpha-actin.

Venoms have evolved over millions of years into potent cocktails of bioactive peptides and proteins. These compounds can be of great value to the pharmaceutical industry for numerous clinical applications. In this study, a novel proteomic - bioinformatic approach was utilised, where chromatography followed by gel electrophoresis was utilised to separate the venom peptides/proteins of Heterometrus longimanus (Asian black scorpion). Purified peptides were analysed by tandem mass spectrometry, de novo sequenced and then homology matched against known peptides in the Swiss-Prot protein database. Numerous potentially biologically active peptide matches were discovered, and a simple scoring system applied to putatively assign functions to the peptides. As a validation of this approach, the functional composition of the experimentally derived proteome is similar to that of other scorpions, and contains a potent mix of toxins, antimicrobials and ionic channel inhibitors.

Griffithsin (GRFT), a novel anti-HIV protein, was isolated from an aqueous extract of the red alga Griffithsia sp. The 121-amino acid sequence of GRFT has been determined, and biologically active GRFT was subsequently produced by expression of a corresponding DNA sequence in Escherichia coli. Both native and recombinant GRFT displayed potent antiviral activity against laboratory strains and primary isolates of T- and M- tropic HIV-1 with EC50 values ranging from 0.043 to 0.63 nM. GRFT also aborted cell-to-cell fusion and transmission of HIV-1 infection at similar concentrations. High concentrations (e.g. 783 nM) of GRFT were not lethal to any tested host cell types. GRFT blocked CD4-dependent glycoprotein (gp) 120 binding to receptor-expressing cells and bound to viral coat glycoproteins (gp120, gp41, and gp160) in a glycosylation-dependent manner. GRFT preferentially inhibited gp120 binding of the monoclonal antibody (mAb) 2G12, which recognizes a carbohydrate-dependent motif, and the (mAb) 48d, which binds to CD4-induced epitope. In addition, GRFT moderately interfered with the binding of gp120 to sCD4. Further data showed that the binding of GRFT to soluble gp120 was inhibited by the monosaccharides glucose, mannose, and N-acetylglucosamine but not by galactose, xylose, fucose, N-acetylgalactosamine, or sialic acid-containing glycoproteins. Taken together these data suggest that GRFT is a new type of lectin that binds to various viral glycoproteins in a monosaccharide-dependent manner. GRFT could be a potential candidate microbicide to prevent the sexual transmission of HIV and AIDS.