Affinity Mass Spectrometry for Targeted Proteomics
Date Issued
2005
Date
2005
Author(s)
Chen, Shu-Hua
DOI
en-US
Abstract
As the field of proteomics evolves from the discovery phase to validation and application, tools for targeted proteomics with high sensitivity and specificity remain to be developed. In this study, two solid sample supports, surface modified PVDF and surface engineered nanoparticles were employed as baits to achieve the affinity capture process.
Human serum is an important medium where specific proteins could become elevated in patients and serve as important biomarkers in diagnosis. However, human serum is among the most proteome with a wide dynamic range of more than 1012 in protein abundance. The dynamic range imposes an extreme analytical difficulty for clinical diagnosis. In this thesis, a simple and effective method was developed in combination of PVDF-aided immunoaffinity extraction and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to circumvent the difficulties. Based on antibody-antigen interactions, the acute phase proteins can be selectively isolated from human serum. We show that the polyvinylidene fluoride (PVDF) membrane can be an effective MALDI target probe that improved the spot-to-spot variation for protein detection, allowing the feasibility of quantitative analysis by MALDI-TOF MS. After antibody was immobilized on the solid support, affinity extraction of corresponding antigen can be obtained from complex solution without additional purification and desalting. Preliminary result showed the detection limit of PVDF-aided immunoaffinity assay was in the nanomalar concentrations. To illustrate the efficacy of the approach, serum amyloid P component (SAP), serum amyloid A protein (SAA) and c-reactive protein (CRP) were selectively extracted from human serum with no prior fractionation. Interferences from nonspecific retention were minimized through choice of appropriate blocking reagent on the PVDF membrane. The calibration curve for SAP quantitation revealed at least 1order of magnitude with a good linear response. The measured SAP level in healthy subjects was 41.7± 12.7 mg/L consistent with the literature of 35-40 mg/L. In our experiments, we found that the levels of SAP show no significant change in serum between healthy subjects and patients with gastric cancer (39.3±15.8 mg/L), a result that is in good agreement with the result of 2-D gel experiments.
Molecular recognition events involving carbohydrates play an essential role in various types of interactions in immune system. Knowledge of such interactions can provide new insights in the development of new therapeutic agents for infection treatment. In the second part of this thesis, we report a new approach, nanoprobe-based affinity mass spectrometry (NBAMS), using ligand encapsulated gold nanoparticle (AuNP) as baits for the separation and enrichment of targeted proteins with enhanced detection, protein characterization, and epitope mapping by MALDI-TOF MS. A bioactive and biocompatible nanoprobe was constructed with 4 nm AuNP, which was covalently attached with galactose (g-AuNP). Simultaneous enrichment and isolation of Pseudomonas aeruginosa lectin I (PA-IL) from a mixture without interfere from other high abundance protein was achieved by using g-AuNP. A concentration with 10000-fold difference can be detected successfully and current detection limit was in subnanomoler level. Targeted protein identification by peptide mass fingerprinting with 100% sequence coverage confirmed the identity of captured PA-IL. Subsequent mapping of the epitope containing peptides were consistent with the literature where the binding sites were determined by X-ray crystallographic analysis. Finally, de novo sequencing of the epitope-containing peptide was demonstrated with direct MALDI TOF/TOF.
The combination of biologically active probes with mass spectrometry is capable to rapidly and specifically extracted proteins of interest followed by quantitation, identification and epitope mapping. Through the use of simple and effective purification technique of PVDF-aided immunoaffinity assay, quantitative screening of a clinically protein profiling in a biological fluid can be achieved. The flexibility of versatilely functionalized nanoprobe can be easily adapted for ligand-fishing of class-specific proteins by NBAMS technique.
Subjects
親和質譜術
基質輔助雷射脫附游離質譜術
奈米粒子
nanoparticle
MALDI MS
affinity mass spectrometry
SDGs
Type
thesis
File(s)![Thumbnail Image]()
Loading...
Name
ntu-94-R91223054-1.pdf
Size
23.31 KB
Format
Adobe PDF
Checksum
(MD5):2fd04a0bf5fd7d67a98a9cb7df3b2676