Accuracy of diagnosis among clinical malaria patients: comparing microscopy, RDT and a highly sensitive quantitative PCR looking at the implications for submicroscopic infections.

BACKGROUND: The World Health Organization recommends parasitological confirmation of all suspected malaria cases by microscopy or rapid diagnostic tests (RDTs) before treatment. These conventional tools are widely used for point-of-care diagnosis in spite of their poor sensitivity at low parasite density. Previous studies in Ghana have compared microscopy and RDT using standard 18S rRNA PCR as reference with varying outcomes. However, how these conventional tools compare with ultrasensitive varATS qPCR has not been studied. This study, therefore, sought to investigate the clinical performance of microscopy and RDT assuming highly sensitive varATS qPCR as gold standard. METHODS: 1040 suspected malaria patients were recruited from two primary health care centers in the Ashanti Region of Ghana and tested for malaria by microscopy, RDT, and varATS qPCR. The sensitivity, specificity, and predictive values were assessed using varATS qPCR as gold standard. RESULTS: Parasite prevalence was 17.5%, 24.5%, and 42.1% by microscopy, RDT, and varATS qPCR respectively. Using varATS qPCR as the standard, RDT was more sensitive (55.7% vs 39.3%), equally specific (98.2% vs 98.3%), and reported higher positive (95.7% vs 94.5%) and negative predictive values (75.3% vs 69.0%) than microscopy. Consequently, RDT recorded better diagnostic agreement (kappa = 0.571) with varATS qPCR than microscopy (kappa = 0.409) for clinical detection of malaria. CONCLUSIONS: RDT outperformed microscopy for the diagnosis of Plasmodium falciparum malaria in the study. However, both tests missed over 40% of infections that were detected by varATS qPCR. Novel tools are needed to ensure prompt diagnosis of all clinical malaria cases.

Rapid quantification of insulin degludec by immunopurification combined with liquid chromatography high-resolution mass spectrometry.

Insulin degludec is an ultra-long-acting insulin analogue that is increasingly being used in diabetes due to its favourable efficacy and safety profile. Thus, there is an increasing demand for a reliable and specific analytical method to quantify insulin degludec for research, pharmaceutical industry and clinical applications. We developed and validated an automated, high-throughput method for quantification of insulin degludec in human blood samples across the expected clinical range combining immunopurification with high-resolution mass spectrometry. Validation was performed according to the requirements of the US Food and Drug Administration. The method satisfyingly met the following parameters: lower limit of quantification (120 pM), linearity, accuracy (error < 5%), precision (CV < 7.7%), selectivity, carry-over, recovery (89.7-97.2%), stability and performance in the presence of other insulin analogues. The method was successfully applied to clinical samples of patients treated with insulin degludec showing a good correlation with the administered dose (r2 = 0.78). High usability of the method is supported by the small specimen volume, automated sample processing and short analysis time. In conclusion, this reliable, easy-to-use and specific mass spectrometric insulin degludec assay offers great promise to address the current unmet need for standardized insulin analytics in academic and industrial research. Graphical Abstract.

CTL recognition of a novel HLA-A*0201-binding peptide derived from glioblastoma multiforme tumor cells.

Genetic instability of tumor cells can result in translation of proteins that are out of frame, resulting in expression of neopeptides. These neopeptides are not self-proteins and therefore should be immunogenic. By eluting peptides from human glioblastoma multiforme (GBM) tumor cell surfaces and subjecting them to tandem mass spectrometry, we identified a novel peptide (KLWGLTPKVTPS) corresponding to a frameshift in the 3' beta-hydroxysteroid dehydrogenase type 7 (HSD3B7) gene. HLA-binding algorithms predicted that a 9-amino acid sequence embedded in this peptide would bind to HLA-A*0201. We confirmed this prediction using an HLA-A*0201 refolding assay followed by live cell relative affinity assays, but also showed that the 12-mer binds to HLA-A*0201. Based on the 9-mer sequence, optimized peptide ligands (OPL) were designed and tested for their affinities to HLA-A*0201 and their abilities to elicit anti-peptide and CTL capable of killing GBM in vitro. Wild-type peptides as well as OPL induced anti-peptide CTL as measured by IFN-γ ELISPOTS. These CTL also killed GBM tumor cells in chromium-51 release assays. This study reports a new CTL target in GBM and further substantiates the concept that rational design and testing of multiple peptides for the same T-cell epitope elicits a broader response among different individuals than single peptide immunization.

Structure of the preamyloid dimer of beta-2-microglobulin from covalent labeling and mass spectrometry.

Beta-2-microglobulin (beta2m) self-associates into fibrillar amyloid deposits in the musculoskeletal system of patients undergoing hemodialysis treatment. Previous studies have shown that stoichiometric amounts of Cu(II) at near physiological conditions can cause beta2m to organize into native-like dimers prior to forming amyloid fibrils. Here, we report the results from selective covalent labeling reactions combined with mass spectrometry that provide insight into the amino acid residues that mediate dimer formation in the wild-type protein. Using three complementary covalent labeling reagents, we find that the dimer interface is formed by the antiparallel stacking of ABED beta-sheets from two beta2m monomers. In addition, our data clearly indicate that a dimer interface involving the interactions of D-D strands from separate protein units as seen in the recent crystal structures of two mutant beta2m oligomers is unlikely.

Analysis of the plasma peptidome from pancreas cancer patients connects a peptide in plasma to overexpression of the parent protein in tumors.

Blood circulates through nearly every organ including tumors. Therefore, plasma is a logical source to search for tumor-derived proteins and peptides. The challenge with plasma is that it is a complex bodily fluid composed of high concentrations of normal host proteins that obscure identification of tumor-derived molecules. To simplify plasma, we examined a low molecular weight (LMW) fraction (plasma peptidome) using liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods. In the plasma peptidome of patients with ductal adenocarcinoma of the pancreas (DAP), a prominent peptide was identified from the QSOX1 parent protein. This peptide is stable in whole blood over 24 h and was present in 16 of 23 DAP patients and 4 of 5 patients with intraductal papillary mucinous neoplasm (IPMN). QSOX1 peptides were never identified in the plasma peptidome from 42 normal healthy donors using the same methods. Immunohistochemical staining of DAP tissue sections with anti-QSOX1 antibody shows overexpression of QSOX1 in tumor but not in adjacent stroma or normal ducts. Three of four pancreas tumor cell lines also express QSOX1 protein by Western blot analysis. This is the first report of QSOX1 peptides in plasma from DAP patients and makes the rare connection between a peptide in plasma from cancer patients and overexpression of the parent protein in tumors.

Development and validation of a targeted affinity-enrichment and LC-MS/MS proteomics approach for the therapeutic monitoring of adalimumab.

BACKGROUND: The anti-tumor necrosis factor alpha (TNFα) therapeutic monoclonal antibodies (mAbs), such as adalimumab, are widely used in the treatment of rheumatoid arthritis, inflammatory bowel diseases, and other auto-immune diseases. The administration of adalimumab can elicit the immune responses from some patients, resulting in the formation of anti-drug antibodies (ADAbs). The ADAbs can diminish the therapeutic effects of adalimumab by neutralizing the TNFα binding site or increasing its clearance from circulation. METHODS: To effectively monitor the therapeutic concentrations of adalimumab, we developed and validated a targeted quantitative proteomic assay to determine the circulating concentrations of adalimumab. Since drug effects can be attenuated by ADAbs, the method adopted an affinity-enrichment step to selectively quantify the bioavailable forms of adalimumab in patient serum samples. RESULTS: The performance of the LC-MS/MS based assay provides the analytical measuring range and precisions applicable for the therapeutic monitoring of adalimumab. It also provides comparable results to a cell-based activity assay when evaluating patient samples with different concentrations of adalimumab. CONCLUSION: Our assay can quantify both sub-therapeutic and therapeutic concentrations of bioavailable adalimumab in patient serum samples. This assay design provides an alternative to isotope-labeled peptides approach currently adopted in targeted proteomics methods.

Quiescin sulfhydryl oxidase 1 promotes invasion of pancreatic tumor cells mediated by matrix metalloproteinases.

Quiescin sulfhydryl oxidase 1 (QSOX1) oxidizes sulfhydryl groups to form disulfide bonds in proteins. We previously mapped a peptide in plasma from pancreatic ductal adenocarcinoma (PDA) patients back to an overexpressed QSOX1 parent protein. In addition to overexpression in pancreatic cancer cell lines, 29 of 37 patients diagnosed with PDA expressed QSOX1 protein in tumor cells, but QSOX1 was not detected in normal adjacent tissues or in a transformed, but nontumorigenic cell line. To begin to evaluate the advantage QSOX1 might provide to tumors, we suppressed QSOX1 protein expression using short hairpin (sh) RNA in two pancreatic cancer cell lines. Growth, cell cycle, apoptosis, invasion, and matrix metalloproteinase (MMP) activity were evaluated. QSOX1 shRNA suppressed both short and long isoforms of the protein, showing a significant effect on cell growth, cell cycle, and apoptosis. However, QSOX1 shRNA dramatically inhibited the abilities of BxPC-3 and Panc-1 pancreatic tumor cells to invade through Matrigel in a modified Boyden chamber assay. Mechanistically, gelatin zymography indicated that QSOX1 plays an important role in activation of MMP-2 and MMP-9. Taken together, our results suggest that the mechanism of QSOX1-mediated tumor cell invasion is by activation of MMP-2 and MMP-9.

Proteomic identification of an MHC-binding peptidome from pancreas and breast cancer cell lines.

Peptides bound to cell surface MHC class I molecules allow the immune system to recognize intracellular pathogens and tumor-derived peptides. Our goal was to learn what the immune system "sees" on the surfaces of tumor cells by acid-eluting peptides from HLA molecules for extended time periods. We determined how long peptides would continue to elute over time from a pancreatic tumor cell line, Panc-1, and a breast cancer cell line, MCF-7, at pH 3.0 in citrate buffer while monitoring viability. Both cell lines demonstrated greater than 90% viability after 25min at pH 3.0. Panc-1 remained >90% intact after 45min at pH 3.0. Acid eluted peptide sequences were identified using LC-MS/MS and searching the NCBI refseq database. The total number of peptides eluted peaked between 40 and 45min for Panc-1, but continued to increase over time from MCF-7. A total of 131 peptides were identified from Panc-1 while 101 peptides were identified from MCF-7 elutions. Two classes of peptides were eluted: (1) 8-10 amino acid peptides fitting the HLA-binding motifs of each cell line, and (2) peptides longer than 10 amino acids containing HLA-binding motifs of each cell line. W6/32 antibody affinity purification of intact MHC molecules after papain cleavage of MHC class I from tumor cell surfaces also indicated that peptides longer than 10 amino acids bind to class I proteins. A peptide-MHC-refolding assay further substantiated the binding of longer peptides to HLA-A*0201. Our findings provide sequences and gene names of peptides presented by MHC class I molecules from common pancreas and breast cancer cell lines. We utilized a novel refolding assay to demonstrate that peptides longer than the canonical 8-10 amino acids commonly bind in MHC class I cell surface molecules.

Cu(II) organizes beta-2-microglobulin oligomers but is released upon amyloid formation.

beta-2-Microglobulin (beta2m) is deposited as amyloid fibrils in the bones and joints of patients undergoing long-term dialysis treatment as a result of kidney failure. Previous work has shown that biologically relevant amounts of Cu(II) can cause beta2m to be converted to amyloid fibrils under physiological conditions in vitro. In this work, dynamic light scattering, mass spectrometry, and size-exclusion chromatography are used to characterize the role that Cu plays in the formation of oligomeric intermediates that precede fibril formation. Cu(II) is found to be necessary for the stability of the dimer and an initial form of the tetramer. The initially formed tetramer then undergoes a structural change to a state that no longer binds Cu(II) before progressing to a hexameric state. Based on these results, we propose that the lag phase associated with beta2m fibril formation is partially accounted for by the structural transition of the tetramer that results in Cu(II) loss. Consistent with this observation is the determination that the mature beta2m amyloid fibrils do not contain Cu. Thus, Cu(II) appears to play a catalytic role by enabling the organization of the necessary oligomeric intermediates that precede beta2m amyloid formation.