Detection of serum M-protein in acetonitrile precipitates by MALDI-TOF mass spectrometry: A novel, low-cost methodology.

BACKGROUND: Several studies have demonstrated the analytical sensitivity of MALDI-TOF mass spectrometry (MALDI-TOF MS) by immunoenrichment for M-protein analysis. We report the results of a novel, low-cost, reagent-based extraction process using acetonitrile (ACN) precipitation to enrich for κ and λ light chains which can be analysed by MALDI-TOF MS. METHODS: Institutional Ethics committee approval was obtained. Serum samples from patients with monoclonal gammopathy of undetermined significance (MGUS), multiple myeloma (MM), plasmacytoma, AL amyloidosis and Waldenström macroglobulinemia (WM) underwent ACN precipitation. The images obtained were overlaid on apparently healthy donor serum samples to confirm the presence of M-protein. A sample was considered positive for M-protein if there was a sharp or broad peak within the κ or λ mass/charge (m/z) range: m/z- [M + 2H]2+: 11,550-12,300 Da and λ m/z- [M + 2H]2+: 11,100-11,500 Da. Images were acquired at a m/z range of 10,000-29,000 Da. Corresponding serum protein electrophoresis (SPEP), serum immunofixation electrophoresis (IFE) and serum free light chain (sFLC) assay by nephelometry were performed for all the samples. RESULTS: Two-hundred-and-two serum samples were included in the study: MM- 184 (91%); AL amyloidosis- 2 (1%); plasmacytoma- 8 (4%); MGUS- 6 (3%) and WM- 2 (1%). All the SPEP positive samples were identified by MALDI-TOF MS. Out of 179 samples positive for M-protein by IFE, MALDI-TOF MS was positive in 176 samples (98%). Compared to IFE, the sensitivity and specificity of M-protein identification by MALDI-TOF MS were 98.3% and 52.2%, respectively. CONCLUSIONS: This study demonstrates the feasibility of qualitatively identifying M-protein without the need for antibody-based immunoenrichment, making the technique cost-effective.

Combination of IDO1high and CCL19low expression in the tumor tissue reduces survival in HPV positive cervical cancer.

The over expression of Indoleamine 2, 3-Dioxygenase (IDO1), an immune checkpoint inhibitor, is well known in cervical cancer. However, its association with chemokine signals promoting cellular infiltration in the cervical tumor microenvironment, is unknown. In the current study, we evaluated the expression and enzymatic activity of IDO1. We also profiled the expression of chemokine ligand-receptors- CCR4-CCL22, CXCR3-CXCL10, CXCR4-CXCL12, and CCR7-CCL19 using immunohistochemistry (IHC), and studied their association with IDO1, statistically. After getting an informed consent, punch biopsy samples were obtained from 105 patients diagnosed with cervical cancer. HPV typing by Sanger sequencing, realtime PCR for quantifying IDO1 mRNA expression, HPLC for determining the K/T ratio and IHC for all the above chemokine receptor-ligand pairs along with IDO1 were performed. We found a significant increase in the expression of IDO1 and K/T levels in early and locally advanced stages when compared to Stage IV disease. Among the chemokine ligand -receptor pairs profiled, we found that high CCL19 marker expression was a good prognostic indicator of patients' disease-free (p = 0.013) and overall survival (p = 0.043). Although we could not identify IDO1 as an independent prognostic factor, we found that high levels of IDO1 expression may further reduce survival outcomes in patients with low CCL19 expression. This could be vital for designing immuno therapeutic interventions targeting IDO1.

Expression and affinity purification of recombinant mammalian mitochondrial ribosomal small subunit (MRPS) proteins and protein-protein interaction analysis indicate putative role in tumourigenic cellular processes.

Mitochondrial ribosomal small subunit (MRPS) group of proteins is structural constituents of the small subunit of mitoribosomes involved in translation. Recent studies indicate role in tumourigenic process, however, unlike cytosolic ribosomal proteins, knowledge on the role of MRPS proteins in alternate cellular processes is very limited. Mapping protein-protein interactions (PPIs) onto known cellular processes can be a valuable tool to identify novel protein functions. In this study, to identify PPIs of MRPS proteins, we have constructed 31 glutathione-S-transferase (GST)/MRPS fusion clones. GST/MRPS fusion proteins were confirmed by MALDI-TOF analysis. GST pull-downs were performed using eight GST/MRPS proteins (MRPS9, MRPS10, MRPS11, MRPS18B, MRPS31, MRPS33, MRPS38 and MRPS39), GST alone as pull-down control and HEK293 cell lysate as the source for anchor proteins followed by nLC/MS/MS analysis and probable PPIs of eight MRPS proteins were identified. Three PPIs from GST pull-downs and interaction between six MRPS proteins and p53 previously reported in PPI database were validated. The PPI network analysis revealed putative role in cellular processes with implications for tumourigenesis. Gene expression screening of a cancer cell line panel indicated overexpression of MRPS10 and MRPS31 in breast cancer. Co-expression module identification tool analysis of breast cancer gene expression and MRPS10 and MRPS31 PPIs revealed putative role for PPI with acyl-CoA dehydrogenase in fatty acid oxidation process regulated by brain-derived neurotrophic factor signalling pathway.