Integrative proteome analysis of bone marrow interstitial fluid and serum reveals candidate signature for acute myeloid leukemia.

Acute myeloid leukemia (AML) is an aggressive form of blood cancer and clinically highly heterogeneous characterized by the accumulation of clonally proliferative immature precursors of myeloid lineage leading to bone marrow failure. Although, the current diagnostic methods for AML consist of cytogenetic and molecular assessment, there is a need for new markers that can serve as useful candidates in diagnosis, prognosis and understanding the pathophysiology of the disease. This study involves the investigation of alterations in the bone marrow interstitial fluid and serum proteome of AML patients compared to controls using label-free quantitative proteomic approach. A total of 201 differentially abundant proteins were identified in AML BMIF, while in the case of serum 123 differentially abundant proteins were identified. The bioinformatics analysis performed using IPA revealed several altered pathways including FAK signalling, IL-12 signalling and production of macrophages etc. Verification experiments were performed in a fresh independent cohort of samples using MRM assays led to the identification of a panel of three proteins viz., PPBP, APOH, ENOA which were further validated in a new cohort of serum samples by ELISA. The three-protein panel could be helpful in the diagnosis, prognosis and understanding of the pathophysiology of AML in the future. BIOLOGICAL SIGNIFICANCE: Acute Myeloid Leukemia (AML) is a type haematological malignancy which constitute one third of total leukemias and it is the most common acute leukemia in adults. In the current clinical practice, the evaluation of diagnosis and progression of AML is largely based on morphologic, immunophenotypic, cytogenetic and molecular assessment. There is a need for new markers/signatures which can serve as useful candidates in diagnosis and prognosis. The present study aims to identify and validate candidate biosignature for AML which can be useful in diagnosis, prognosis and understand the pathophysiology of the disease. Here, we identified 201 altered proteins in AML BMIF and 123 in serum. Among these altered proteins, a set of three proteins viz., pro-platelet basic protein (CXCL7), enolase 1 (ENO1) and beta-2-glycoprotein 1 (APOH) were significantly increased in AML BMIF and serum suggest that this panel of proteins could help in future AML disease management and thereby improving the survival expectancy of AML patients.

A single step and rapid protein extraction protocol developed for cell lines and tissues: Compatible for gel based and gel free proteomic approaches.

Proteins are crucial research molecules in modern biology. Almost every biological research area needs protein-based assays to answer the research questions. The study of the total protein content of a biological sample known as Proteomics, is one of the highly rated qualitative and quantitative approach to address numerous biological problems including clinical research. The key step to successfully generate high quality proteomics data is the efficient extraction of proteins from biological samples. Although different methods are in use for protein extraction from a wide variety of samples, however, because of their prolonged protocol and multiple steps involved, final protein yield is sacrificed. Here, we have shown the development of a simple single step method for extraction of proteins from mammalian cell lines as well as tissue samples in an effective and reproducible manner. This method is based on lysis of samples directly in a modified lysis buffer without CHAPS (7 M Urea, 2 M Thiourea, and 10 mM Tris-Cl; pH 8.5) that is compatible with gel based and gel free approaches. This developed protocol is reliable and should be useful for a wide range of proteomic studies involving various biological samples.

Investigation of altered urinary metabolomic profiles of invasive ductal carcinoma of breast using targeted and untargeted approaches.

INTRODUCTION: Invasive ductal carcinoma (IDC) is a type of breast cancer, usually detected in advanced stages due to its asymptomatic nature which ultimately leads to low survival rate. Identification of urinary metabolic adaptations induced by IDC to understand the disease pathophysiology and monitor therapy response would be a helpful approach in clinical settings. Moreover, its non-invasive and cost effective strategy better suited to minimize apprehension among high risk population. OBJECTIVE: This study aims toward investigating the urinary metabolic alterations of IDC by targeted (LC-MRM/MS) and untargeted (GC-MS) approaches for the better understanding of the disease pathophysiology and monitoring therapy response. METHODS: Urinary metabolic alterations of IDC subjects (63) and control subjects (63) were explored by targeted (LC-MRM/MS) and untargeted (GC-MS) approaches. IDC specific urinary metabolomics signature was extracted by applying both univariate and multivariate statistical tools. RESULTS: Statistical analysis identified 39 urinary metabolites with the highest contribution to metabolomic alterations specific to IDC. Out of which, 19 metabolites were identified from targeted LC-MRM/MS analysis, while 20 were identified from the untargeted GC-MS analysis. Receiver operator characteristic (ROC) curve analysis evidenced 6 most discriminatory metabolites from each type of approach that could differentiate between IDC subjects and controls with higher sensitivity and specificity. Furthermore, metabolic pathway analysis depicted several dysregulated pathways in IDC including sugar, amino acid, nucleotide metabolism, TCA cycle etc. CONCLUSIONS: Overall, this study provides valuable inputs regarding altered urinary metabolites which improved our knowledge on urinary metabolomic alterations induced by IDC. Moreover, this study identified several dysregulated metabolic pathways which offer further insight into the disease pathophysiology.