No abstract

Glioblastoma multiforme (GBM) is the most common and aggressive type of primary malignant tumor of the central nervous system. We have carried out a deep analysis of the secretome of a rapidly proliferating and tumorigenic cell line HNGC-2, representing GBM, in an effort to identify proteins, which may be targeted in the plasma of GBM patients as markers for diagnosis and disease surveillance. Prefractionation of the proteins from the conditioned medium of HNGC-2 cells in SDS gels followed by LC-MS/MS analysis using an ESI-IT mass spectrometer (LTQ) led to a total of 996 protein identifications with ≥2 peptides each. Of them, 664 proteins were observed in the transcriptome of HNGC-2 cells. The dataset of 996 proteins was mapped to important functional groups, such as cellular assembly and organisation, DNA recombination and repair, and other classes. Actin cytoskeleton signalling, phosphatidyl inositol 3 kinase (PI3K/AKT) and integrin linked kinase (ILK) signalling pathways were seen as enriched pathways. Comparisons with the published secretome of cell lines from 12 different cancers, including GBM, revealed that 348 proteins shared a commonality with a secretome of at least one other cell line, 321 of which were found to contain signal sequences or transmembrane domains and 335 could be linked to a plasma membrane or extracellular localization. Through intergration of this data we arrived at a non-redundant list of 597 protein identifications with the potential for secretion either by classical secretory pathways or by non-secretory processes; 233 of them have been detected in cerebrospinal fluid or plasma as per the published literature, and 172 have been implicated in GBM or other cancers. The HNGC-2 secretome dataset could serve as a useful resource for designing a targeted investigation of GBM biomarkers in plasma.

Analysis of any mammalian plasma proteome is a challenge, particularly by mass spectrometry, due to the presence of albumin and other abundant proteins which can mask the detection of low abundant proteins. As detection of human plasma proteins is valuable in diagnostics, exploring various workflows with minimal fractionation prior to mass spectral analysis, is required in order to study population diversity involving analysis in a large cohort of samples. Here, we used ‘reference plasma sample’, a pool of plasma from 10 healthy individuals from Indian population in the age group of 25–60 yrs including 5 males and 5 females. The 14 abundant proteins were immunodepleted from plasma and then evaluated by three different workflows for proteome analysis using a nanoflow reverse phase liquid chromatography system coupled to a LTQ Orbitrap Velos mass spectrometer. The analysis of reference plasma sample a) without prefractionation, b) after prefractionation at peptide level by strong cation exchange chromatography and c) after prefractionation at protein level by sodium dodecyl sulfate polyacrylamide gel electrophoresis, led to the identification of 194, 251 and 342 proteins respectively. Together, a comprehensive dataset of 517 unique proteins was achieved from all the three workflows, including 271 proteins with high confidence identified by≥2 unique peptides in any of the workflows or identified by single peptide in any of the two workflows. A total of 70 proteins were common in all the three workflows. Some of the proteins were unique to our study and could be specific to Indian population. The high-confidence dataset obtained from our study may be useful for studying the population diversity, in discovery and validation process for biomarker identification.

Abstract:Lack of crystal structure data of folate binding proteins has left so many questions unanswered (for example, important residues in active site, binding domain, important amino acid residues involved in interactions between ligand and receptor). With sequence alignment and PROSITE motif identification, we attempted to answer evolutionarily significant residues that are of functional importance for ligand binding and that form catalytic sites. We have analyzed 46 different FRs and FBP sequences of various organisms obtained from Genbank. Multiple sequence alignment identified 44 highly conserved identical amino acid residues with 10 cysteine residues and 12 motifs including ECSPNLGPW (which might help in the structural stability of FR).