Proteins In Small Volumes Research Articles

Proteomic biomarkers of non-small cell lung cancer patients.

Lung cancer is a disease with a very low 5-year survival rate (6-13%) worldwide. The most frequently diagnosed histological type of this cancer is non-small cell lung cancer (NSCLC). Poor prognosis for lung cancer - including NSCLC - is mainly related to the fact that patients are diagnosed in the advanced stages of the disease. The aim of this study is to summarize data that concerns new directions of research regarding diagnostic biomarkers that could be used to support the routine diagnosis of this cancer. In recent years, proteomic analysis has become an important tool for cancer biology research, complementing genetic analysis. Among the numerous methods of proteomic analysis, mass spectrometry techniques enable the extremely accurate qualitative and quantitative identification of hundreds of proteins in small volumes of various biological samples. Such analyses may soon become the basis of improvement in lung cancer diagnostic procedures. This study presents the latest reports in proteomic research concerning the diagnosis of NSCLC. New potential proteomic biomarkers, whose presence indicates the development of a neoplastic process at an early stage, are presented. We describe biomarkers whose altered expression levels correlate with different stages of cancer. We also present protein biomarkers that help differentiate NSCLC subtypes. In the clinical workup of NSCLC patients, it is important not only to make an early diagnosis, but also to monitor the development of the neoplastic disease. Considering this fact, we also present examples of biomarkers whose abnormal expression may indicate a high risk of metastasis to the lymph nodes. This paper also emphasizes the need to conduct further research that would confirm the usefulness of the described biomarkers in clinical practice.

Comparison of the performance of two affinity depletion spin filters for quantitative proteomics of CSF: Evaluation of sensitivity and reproducibility of CSF analysis using GeLC‐MS/MS and spectral counting

For biomarker discovery in cerebrospinal fluid (CSF), removal of major serum proteins is advantageous as more CSF proteins including brain-derived proteins can be identified. Our goal was to create a reproducible discovery workflow with acceptable throughput that can identify 500-1000 CSF proteins in small volumes of CSF. In this study, we compared the performance of two multi-affinity depletion methods in spin filter format: MARS Human 14 and Seppro-IgY-14. To this end, we analyzed depleted and bound CSF fractions isolated from 0.5 mL aliquots of the same CSF sample (n=3 per depletion method) by label-free GeLC-MS/MS-based proteomics and normalized spectral counting. The whole CSF dataset contained 884 proteins identified at high confidence. Depletion spin filter performance was assessed in terms of sensitivity and reproducibility of the CSF analysis. MARS and IgY-14 spin filters yielded comparable reproducibility of protein identification (71-74%) and quantification (CV 17-18%) but a significant difference in the total number of identified CSF proteins (767 and 703 proteins, respectively). The MARS filter compared to IgY-14 filter provides a CSF analysis with enhanced proteome coverage. We anticipate that this enhanced sensitivity will facilitate biomarker discovery in early stages of cancer or neurological disease.

Integration of a membrane-based desalting step in a microfabricated disposable polymer injector for mass spectrometric protein analysis.

A simple desalting procedure for the coupling of a polymer microchip injector to mass spectrometry is proposed. The overall process is based on the adsorption of proteins on a poly(vinylidene difluoride) (PVDF) membrane, which are then directly eluted in the spraying solution. This microchip-based approach has been successfully applied to small drugs, peptides and proteins originally diluted in phosphate-buffered saline (PBS). Moreover, when eluting the retained proteins in small volumes, a preconcentration is obtained. The combination of single-use, mass-produceable, low-sample-consumption, easy-to-automate, miniaturized polymer injectors with easy-to-handle solution-exchange membranes makes this system particularly amenable to screening applications.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis of proteins in human cerebrospinal fluid.

Matrix-assisted laser desorption/ionization time-of-flight mass spectra of proteins in cerebrospinal fluid analyzed without prior purification are presented. Less than 100 fmol amounts of proteins in the 10,000 to 20,000 u mass range and linked to human disease (multiple sclerosis, Alzheimer's disease, and stroke) were detected in a complex mixture of proteins and peptides, in the presence of high concentrations of salts, lipids and free amino acids. The mass resolution was sufficient to distinguish between the non-hydroxylated and hydroxylated forms of a 13,400 u protein. Simple fractionation of the cerebrospinal fluid using microbore-reversed phase high performance liquid chromatography improved signal-to-noise ratios in the mass spectra. High-accuracy peptide mass mapping and database searching were utilized to confirm the identity of several proteins. The presented results show that matrix-assisted laser desorption/ionization time-of-flight mass spectrometry could be used as a tool to perform rapid screening of chemically altered proteins in small volumes of biological fluids.

A rapid method of concentrating proteins in small volumes with high recovery using sephadex G-25

A method for concentrating small (10 to 500 μl) volumes of protein solutions by mixing in a microcentrifuge tube with measured volumes of dry Sephadex G-25 is described. The tube is centrifuged, and the protein solution concentrated up to fivefold passes through a small hole in the base of the tube and is collected. Recovery of protein is 90–95%.

Sequential elution of proteins in small volumes by preparative polyacrylamide gel electrophoresis

A simple flexible method for separation of proteins by polyacrylamide gel electrophoresis and sequential elution into dialysis bags has been devised. The system was applied to isolation of three glycoproteins from the peritoneal fluid of mice bearing Ehrlich ascites tumor.

Densitometric determination of proteins in small volumes

An analytical method of quantitative determination of proteins in small volumes is described. The method is based upon the densitometric evaluation of colour tests in agar-gel. The quantity of proteins is determined by graphical integration of densitometric curves and, in the arrangement described, it is possible to assay proteins in volumes of the order 10−3 ml.