Application of MALDI-TOF for biomarker determination

Application of MALDI-TOF for biomarker determination: A straightforward and reproducible tool for rapidly scanning serum proteome, detecting and distinguishing hundreds of low molecular weight proteins and peptides in seconds.

By Alicia Prieto, PhD, Chromatography Service, Centro de Investigaciones Biológicas,Consejo Superior de Investigaciones Cientificas Madrid, Spain:aliprieto@cib.csic.es

One of the most active MALDI-TOF application areas is in the analysis of disease-specific biomarker peptides and low abundance pro teins in blood, urine, and other biological fluids. These molecules are used as diseasespecific biomarkers. It is the aim of this type of research to use these biomarkers to aid in the diagnosis of disease and in the evaluation of its severity and progression.

Of particular interest is the study of the serum proteome, a complex mixture comprising mainly highly abundant proteins, such as albumin and other carrier proteins, and proteins that originate from circulating blood cells. The population of proteins that enters the blood from the surrounding tissue constitutes a minor fraction, but it may contain most of the undiscovered biomarkers. MALDI-TOF has demonstrated it can be a straightforward and reproducible tool for rapidly scanning these samples, detecting and distinguishing hundreds of low molecular weight proteins and peptides in seconds.

The MALDI protocol first involves the capture and enrichment of peptides using magnetic beads or some other chromatographic method. In the second step a portion of the enriched sample is mixed with a matrix like α-cyano-4-hydroxycinnamic acid. Next, the mixture is spotted onto a target and allowed to dry at room temperature. The final step is the desorption of bulk portions of the solid sample by a short pulse of laser light. The matrix absorbs the laser energy, causing codesorption of the analyte and promoting analyte ionization. Liberated ions from the sample are swept by vacuum into the mass analyzer.

The resulting spectra can be recorded in the linear positive ion mode with external calibration in the range of analysis (1-15 kDa) using a mixture of peptide/protein standards. Up to 400 peptides can be detected and characterized in this mass range. This approach has been used in studies focused on the detection of biomarkers for asthma, acute lymphatic leukemia and brain tumors, among many others. In each case, specific profiles of peptides have been observed that can be used to predict if the sample is normal or diseased.

Two applications on protein identification by MALDITOF are shown below. The first one refers to identification of a bacterial protein (A), and the second one to porcine seminal protein from boar (PSP-II, B).

Sigma-Aldrich offers many MALDI products (matrices, validation and calibration sets), including those for analysis of peptides, proteins, oligonucleotides, syn thetic polymers, organic molecules, carbohydrates, dendrimers.

Application: MALDI-TOF analysis of tryptic digests of (A) un identified bacterial protein and (B) PSP-II. The laser desorption/ ionisation experiments were performed on a BIFLEX III time-of-flight instrument (Bruker-Franzen Analytik, Bremen, Germany) operated in the positive mode. A saturated solution of CCA in acetonitrile:water (1:2) with 0.1 % TFA was used as the matrix. Samples were analysed in the reflectron mode, and typically 50-100 laser shots were summed into a single mass spectrum. External calibration was performed, using peptide calibration standards. Equal volumes (0.5 micro liters) of the sample solution and the matrix were spotted on the target and air-dried at room temperature.

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