A magnetic bead‐based serum proteomic fingerprinting method for parallel analytical analysis and micropreparative purification

Abstract

ProteinChip surface-enhanced laser desorption/ionization technology and magnetic beads-based ClinProt system are commonly used for semi-quantitative profiling of plasma proteome in biomarker discovery. Unfortunately, the proteins/peptides detected by MS are non-recoverable. To obtain the protein identity of a MS peak, additional time-consuming and material-consuming purification steps have to be done. In this study, we developed a magnetic beads-based proteomic fingerprinting method that allowed semi-quantitative proteomic profiling and micropreparative purification of the profiled proteins in parallel. The use of different chromatographic magnetic beads allowed us to obtain different proteomic profiles, which were comparable to those obtained by the ProteinChip surface-enhanced laser desorption/ionization technology. Our assays were semi-quantitative. The normalized peak intensity was proportional to concentration measured by immunoassay. Both intra-assay and inter-assay coefficients of variation of the normalized peak intensities were in the range of 4-30%. Our method only required 2 microL of serum or plasma for generating enough proteins for semi-quantitative profiling by MALDI-TOF-MS as well as for gel electrophoresis and subsequent protein identification. The protein peaks and corresponding gel spots could be easily matched by comparing their intensities and masses. Because of its high efficiency and reproducibility, our method has great potentials in clinical research, especially in biomarker discovery.

Exploration of Serum Proteomic Profiling and Diagnostic Model That Differentiate Crohn's Disease and Intestinal Tuberculosis

“…MALDI-TOF-MS technology is a newly developing kind of proteomics research methods in recent years, with a theory that the laser radiates the crystals comprised of sample and matrix, leading to sample ionization, ionized sample fly accelerated through pipes with the effect of electric field, by detecting the flight time to the detector, M/Z is measured. This method, with advantages of low sample consumption, easy operation, high resolution, good repeatability and high sensitivity, can detect and differentiate polypeptides containing diagnostic message and obtain polypeptide biological information with high sensitivity [ 26 – 28 ] . When detecting low abundance proteins with small relative molecular mass, the effects are more apparent.…”

A Comprehensive Peptidome Profiling Technology for the Identification of Early Detection Biomarkers for Lung Adenocarcinoma

“…From this point of view, the previous peptidome profiling technologies, such as SELDI-TOF-MS coupled with ProteinChip arrays or MALDI-TOF-MS analysis of ClinProt magnetic beads-purified samples, covered only limited spectra of serum peptidome. Most of studies utilizing ion-exchange selection or reversed phase extraction of peptidome on ProteinChip arrays [25], [26], [27] or magnetic beads [28], [29] allowed at most 200 peak detections within the mass range 1,000 to 20,000. Meanwhile our peptidome profiling technology consisting of gel-filtration chromatography, custom-made high resolution C18 tip-column, QSTAR-Elite mass spectrometer, and Expressionist proteome server platform analysis enabled us to detect 12,396 non-redundant molecules with charge state of +1 to +10.…”

Preliminary Application of WCX Magnetic Bead-Based Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry in Analyzing the Urine of Renal Clear Cell Carcinoma

“…The molecular weights of many identified significant proteins are below 5 000 Da, while the minimum molecular weight that classical proteomics technique, such as 2D gel electrophoresis, is capable to detect It is simple, rapid, with high purity, and suitable for clinical application. [6][7][8] When the magnetic beads chip is adopted, protein isolation can be repeatedly implemented for times, 9 which results in good consistency, feasibility and credibility.…”