Proteomic Analysis of Whole Blood Using Volumetric Absorptive Microsampling for Precision Medicine Biomarker Studies.

Microsampling of patient blood promises several benefits over conventional phlebotomy practices to facilitate precision medicine studies. These include at-home patient blood collection, supporting telehealth monitoring, minimal postcollection processing, and compatibility with nonrefrigerated transport and storage. However, for proteomic biomarker studies, mass spectrometry of whole blood has generally been avoided in favor of using plasma or serum obtained from venepuncture. We evaluated the use of a volumetric absorptive microsampling (VAMS) device as a sample preparation matrix to enable LC-MS proteomic analyses of dried whole blood. We demonstrated the detection and robust quantitation of up to 1600 proteins from single-shot shotgun-LC-MS analysis of dried whole blood, greatly enhancing proteome depth compared with conventional single-shot LC-MS analyses of undepleted plasma. Some proteins not previously reported in blood were detected using this approach. Various washing reagents were used to demonstrate that proteins can be preferentially removed from VAMS devices prior to downstream analyses. We provide a demonstration that archival frozen blood cell pellets housed under long-term storage (exceeding 5 years) are compatible with VAMS to enable quantitation of potential biomarker proteins from biobank repositories. These demonstrations are important steps in establishing viable analysis workflows to underpin large-scale precision medicine studies. Data are available via ProteomeXchange with the identifier PXD028605.

Proteome Analysis of Whole Blood Collected by Volumetric Absorptive Microsampling.

Proteomic biomarker discovery and analysis from human biofluids using liquid chromatography-mass spectrometry (LC-MS) is an area of intense biomedical research. There is a growing interest to analyze microsampled patient blood specimens as this is potentially more patient-friendly enabling at-home and bedside self-collection of small blood volumes. However, there are limited studies applying LC-MS proteomic analysis of whole blood as it is dominated by red blood cell proteins such as hemoglobin which suppresses the detection of other less abundant proteins. Volumetric absorptive microsampling (VAMS) devices overcome this issue in part by providing a trapping matrix which allows depletion of abundant blood cell proteins through washing, prior to proteolysis and LC-MS. This approach allows the analysis of proteins from erythrocytes, leukocytes, and plasma and leads to deeper proteomic coverage compared to conventional plasma proteomics, increasing the prospects to discover novel biomarker proteins.

Methylation of translation-associated proteins in Saccharomyces cerevisiae: Identification of methylated lysines and their methyltransferases.

This study aimed to identify sites of lysine methylation in Saccharomyces cerevisiae and the associated methyltransferases. Hexapeptide ligand affinity chromatography was used to normalize the abundance levels of proteins in whole cell lysate. MS/MS, in association with antibody-based detection, was then used to identify lysine methylated proteins and the precise sites of modification. Lysine methylation was found on the proteins elongation factor (EF) 1-α, 2, and 3A, as well as ribosomal proteins 40S S18-A/B, 60S L11-A/B, L18-A/B, and L42-A/B. Precise sites were mapped in all cases. Single-gene knockouts of known and putative methyltransferase(s), in association with MS/MS, showed that EF1-α is monomethylated by Efm1 at lysin 30 and dimethylated by See1 at lysine 316. Methyltransferase Rkm1 was found to monomethylate 40S ribosomal protein S18-A/B at lysine 48. Knockout analysis also revealed that putative methyltransferase YBR271W affects the methylation of proteins EF2 and 3A; this was detected by Western blotting and immunodetection. This methyltransferase shows strong interspecies conservation and a tryptophan-containing motif associated with its active site. We suggest that enzyme YBR271W is named EF methyltransferase 2 (Efm2), in line with the recent naming of YHL039W as Efm1.

Immunoproteomic approach to elucidating the pathogenesis of cryptococcosis caused by Cryptococcus gattii.

Cryptococcosis caused by Cryptococcus gattii is a devastating disease of immunocompetent hosts with an incompletely understood pathogenesis. Utilizing an immunoproteomic approach in a naturally occurring koala model of disease, a number of key proteins and pathways are identified in the early and late pathogenesis of cryptococcosis for the first time. In particular, the thioredoxin system appears important in the pathogenesis of cryptococcosis caused by C. gattii VGII.

Micropreparative fractionation of the complexome by blue native continuous elution electrophoresis.

The large-scale analysis of protein complexes is an emerging challenge in the field of proteomics. Currently, there are few methods available for the fractionation of protein complexes that are compatible with downstream proteomic techniques. Here, we describe the technique of blue native continuous elution electrophoresis (BN-CEE). It combines the features of blue native PAGE (BN-PAGE) and continuous elution electrophoresis (CEE), generating liquid-phase fractions of protein complexes of up to 800 kDa. The resulting complexes can be further analysed by BN-PAGE, by SDS-PAGE and/or by MS. This can help define the constituent proteins of many complexes and their stoichiometry. As BN-CEE is also micropreparative, with a capacity to separate milligram quantities of protein complexes, it will assist the study of proteins of lower abundance. In this regard, the acrylamide concentration and elution rate during separation can be controlled to help 'zoom in' on particular high mass regions and thus complexes of interest. We illustrate the utility of the technique in the analysis of Saccharomyces cerevisiae cellular lysate.

Specific non-peroxide antibacterial effect of manuka honey on the Staphylococcus aureus proteome.

Manuka honey, derived from the New Zealand flowering plant Leptospermum scoparium, shows promise as a topical antibacterial agent and effective chronic wound dressing. The aim of this study was to determine the non-peroxide antibacterial effects of this honey on the proteome of the common wound pathogen Staphylococcus aureus. Proteomic analysis was performed on cells treated for a short time with manuka honey compared with the proteome of untreated cells as well as cells treated with a Leptospermum honey sample without antibacterial activity. Treatment with manuka honey resulted in a significant decrease in the bacterial cell growth rate as well as downregulation of ten and upregulation of two proteins. Nine of these proteins were also differentially expressed by cells treated with the inactive Leptospermum honey, but to a lesser degree, and the rate of bacterial growth was not affected. The differentially expressed proteins have roles in ribosomal function, protein synthesis, metabolic processes and transcription. Manuka honey uniquely caused downregulation of two proteins [dihydrolipoamide dehydrogenase (DLD) and elongation factor Tu (EF-Tu)] associated with two of these pathways as well as upregulation of one stress-related protein [cold shock protein C (CspC)]. The proteomic profile following treatment with manuka honey differed from the profiles of other antibacterial agents, indicating a unique mode of action and its potential value as a novel antimicrobial agent.

Proteomic analysis of intra-arterial thrombus secretions reveals a negative association of clusterin and thrombospondin-1 with abdominal aortic aneurysm.

OBJECTIVE: Abdominal aortic aneurysm (AAA) is usually accompanied by the formation of a large volume of intra-luminal thrombus (ILT). ILT-derived proteins have been suggested as circulating markers for AAA. We conducted a proteomic study screening whole and hexapeptide ligand library (HLL) treated ILT explant secretions to identify potential ILT-derived markers for AAA. METHODS: Unfractionated and HLL-treated ILT secretions from 3 AAA patients were analysed in parallel using liquid chromatography tandem mass spectrometry (LC-MS/MS). In silico analyses were employed to identify proteins with biomarker potential. Proteomic findings were validated by measuring serum concentrations of 2 representative ILT proteins in 313 AAA patients and 690 controls. RESULTS: A total of 150 proteins were identified from thrombus conditioned media; HLL treatment enabled the detection of 53 previously unseen polypeptides. Gene ontology analysis revealed high representation of platelet-secreted proteins. Thrombospondin-1 (TSP-1) and clusterin were selected for further assessment. Serum TSP-1 and clusterin were negatively associated with AAA after adjusting for other risk factors. Odds ratio and 95% confidence intervals were 0.62, 0.41-0.94, and 0.50, 0.33-0.75, for men with serum TSP-1 and clusterin in the fourth compared to first quartiles, respectively. CONCLUSION: This proteomic analysis has identified a group of proteins concentrated in AAA ILT. Assessment of circulating concentrations of two representative polypeptides suggests for the first time that the ILT selectively sequesters proteins rather than actively releasing them. Further work is required to assess the mechanisms underpinning this observation and the associated clinical implications.

Development of a Cryptosporidium oocyst assay using an automated fiber optic-based biosensor.

An intestinal protozoan parasite, Cryptosporidium parvum, is a major cause of waterborne gastrointestinal disease worldwide. Detection of Cryptosporidium oocysts in potable water is a high priority for the water treatment industry to reduce potential outbreaks among the consumer populace. Anti-Cryptosporidium oocyst polyclonal and monoclonal antibodies were tested as capture and detection reagents for use in a fiber optic biosensor assay for the detection of Cryptosporidium oocysts. Antibodies were validated using enzyme-linked immunosorbent assays, flow cytometry, Western blotting and fluorescent microscopy. Oocysts could be detected at a concentration of 105 oocysts/ml when the polyclonal antibodies were used as the capture and detection reagents. When oocysts were boiled prior to detection, a ten-fold increase in sensitivity was achieved using the polyclonal antibody. Western blotting and immunofluorescence revealed that both the monoclonal and polyclonal antibodies recognize a large (>300 kDa) molecular weight mucin-like antigen present on the surface of the oocyst wall. The polyclonal antibody also reacted with a small (105 kDa) molecular weight antigen that was present in boiled samples of oocysts. Preliminary steps to design an in-line biosensor assay system have shown that oocysts would have to be concentrated from water samples and heat treated to allow detection by a biosensor assay.

Improved 2-DE of microorganisms after acidic extraction.

2-DE separations of protein extracts sometimes have problems with poor resolution and streaking. This problem is particularly apparent with microorganisms, most notably those with a large cell wall. Here we describe a novel, rapid protocol for the extraction of microorganisms in acidic conditions, leading to increased resolution and 2-D gel quality. The efficiency of the protocol is demonstrated with extracts of bacteria, Escherichia coli and Bacillus subtilis; fungus, Trichoderma harzianum and yeast, Saccharomyces cerevisiae. We also demonstrate using a membrane centrifugal filtration, that large acidic molecules in excess of 100 kDa, probably including cell wall material, are responsible for the separation difficulties. A range of acidic extraction conditions were investigated, and it was found that optimal extraction is achieved using an extraction solution acidified to pH 3 by 80 mM citric acid. These findings have significant implications for the proteomic study of many medically, agriculturally and environmentally significant microorganisms, as the cell walls of these organisms are often considerably more complex than many commonly studied laboratory strains.

Proteomic response of the biological control fungus Trichoderma atroviride to growth on the cell walls of Rhizoctonia solani.

Trichoderma atroviride has a natural ability to parasitise phytopathogenic fungi such as Rhizoctonia solani and Botrytis cinerea, therefore providing an environmentally sound alternative to chemical fungicides in the management of these pathogens. Two-dimensional electrophoresis was used to display cellular protein patterns of T. atroviride (T. harzianum P1) grown on media containing either glucose or R. solani cell walls. Protein profiles were compared to identify T. atroviride proteins up-regulated in the presence of the R. solani cell walls. Twenty-four protein spots were identified using matrix-assisted laser desorption ionisation mass spectrometry, liquid chromatography mass spectrometry and N-terminal sequencing. Identified up-regulated proteins include known fungal cell wall-degrading enzymes such as N-acetyl-beta-D: -glucosaminidase and 42-kDa endochitinase. Three novel proteases of T. atroviride were identified, containing sequence similarity to vacuolar serine protease, vacuolar protease A and a trypsin-like protease from known fungal proteins. Eukaryotic initiation factor 4a, superoxide dismutase and a hypothetical protein from Neurospora crassa were also up-regulated as a response to R. solani cell walls. Several cell wall-degrading enzymes were identified from the T. atroviride culture supernatant, providing further evidence that a cellular response indicative of biological control had occurred.

Fungal proteomics: initial mapping of biological control strain Trichoderma harzianum.

Trichoderma harzianum is a soil-borne filamentous fungus that exhibits biological control properties. T. harzianum can prevent the growth of pathogenic fungi on many types of plant crops, providing a chemically benign alternative to fungicidal agents currently on the market. A proteomic approach was taken to separate and identify proteins from a strain of T. harzianum with well established biocontrol properties. We developed a method of extracting proteins under acidic conditions that increased the solubilisation of alkaline proteins and eliminated acidic cell wall artefacts from micro-organisms in general. Combined with the use of protease inhibitors, this sample preparation method resulted in hundreds of proteins from T. harzianum being extracted and separated by two-dimensional gel electrophoresis. Proteins were identified by a combination of matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry and liquid chromatography mass spectrometry (LC MS/MS). Manual de novo sequencing was conducted to obtain sequence tags on unidentified proteins. A total of 25 protein spots were positively identified from a whole-cell protein reference map of T. harzianum.

Unseen proteome: mining below the tip of the iceberg to find low abundance and membrane proteins.

Abundant and hydrophilic nonmembrane proteins with isoelectric points below pH 8 are the predominant proteins identified in most proteomics projects. In yeast, however, low-abundance proteins make up 80% of the predicted proteome, approximately 50% have pl's above pH 8 and 30% of the yeast ORFs are predicted to encode membrane proteins with at least 1 trans-membrane span. By applying highly solubilizing reagents and isoelectric fractionation to a membrane fraction of yeast we have a purified and identified 780 protein isoforms, representing 323 gene products, including 28% low abundance proteins and 49% membrane or membrane associated proteins. More importantly, considering the frequency and importance of co- and post-translational modifications, the separation of protein isoforms is essential and two-dimensional electrophoresis remains the only technique which offers sufficient resolution to address this at a proteomic level.