A Novel Approach Using MALDI-TOF/TOF Mass Spectrometry and Prestructured Sample Supports (AnchorChip Technology) for Proteomic Profiling and Protein Identification

Abstract

Mass spectrometry (MS)-based proteomic profiling and protein identification has become a powerful tool for the discovery of new disease biomarkers. Among the MS platforms, matrix-assisted laser desorption/ionization time-of-flight/time-of-flight (MALDI-TOF/TOF) MS offers high sample throughput and the flexibility to couple with different off-line sample fractionation techniques. Here, we present a strategy using MALDI-TOF/TOF MS to analyze fractionated human serum samples for proteomic profiling and then identify serum peptides from these proteomic profiles. We achieve the profiling analyses by using different functionalized magnetic beads to enrich specific subsets of serum proteins/peptides based on their absorption to these beads. This step is followed by elution, transfer onto prestructured sample supports (AnchorChip targets), and analysis in a MALDI-TOF/TOF mass spectrometer. Selected serum peptides are then analyzed in the tandem MS (TOF/TOF) mode to generate fragment ions for determination of their amino acid sequences. We have demonstrated that using this approach, proteomic profiling and protein identification can be done in a single MS instrument.

Mass spectrometry of peptides and proteins from human blood

“…The use of ion exchangers such as DEAE followed by C18 produced further identifications of endogenous peptide ladders from more parent proteins compared to C18 alone (Marshall et al, 2003. The use of magnetic beads to capture blood peptides has been examined (de Noo et al, 2005(de Noo et al, , 2006aGatlin-Bunai et al, 2007;Tiss et al, 2007;Leung & Pitts, 2008;Yao et al, 2008;Callesen et al, 2008a;Qiu et al, 2009;Huang et al, 2009c). Packed chromatography resins were observed to outperform the use of loose media collected in the batch mode (Tiss et al, 2007).…”

“…Recently, the use of parent ion separations in the gas phase was shown to increase both the sensitivity and the specificity of LC-MS/MS of blood (Liu et al, 2007c). Modified surfaces for MALDI plates might provide some advantages for the use of affinity reagents (Weinberger, Morris, & Pawlak, 2000;Weinberger et al, 2002;Leung & Pitts, 2008) and provide for rapid analysis (Corr et al, 2006). The robustness and sensitivity of mass spectrometry of blood is only likely to increase in the future, in part due to enhancements in the evolution, separation and capture of the products of ionization (Guzzetta, Thakur, & Mylchreest, 2002;Corr et al, 2006;Liu et al, 2007c;Kelly et al, 2008).…”

Shotgun proteome analysis of honeybee venom using targeted enrichment strategies

“…Prior studies of honeybee venom employing both "bottom-up" and "topdown" strategies led to identification of only six known venom proteins and three unknown protein components, not identified earlier (Matysiak et al, 2011b;Peiren et al, 2005). Although, PAC targets are dedicated for the rapid screening analysis, many reports have shown that application of the classical AnchorChip MALDI plate facilitates better results (Du et al, 2010;Leung and Pitts, 2008;Zhang et al, 2008Zhang et al, , 2007. This plate, according to the manufacturer is preferred for high-throughput MALDI measurements that are performed in unattended, fully automatic mode.…”

Biomarker Characterization by MALDI–TOF/MS

“…Since biomolecular profiling will reflect changes in developmental, physiological, and pathological processes [178][179][180][181][182][183][184][185], several approaches have used bimolecular profiling for valid clinical diagnoses. Multivariate analysis such as principal component analysis (PCA) is often involved in these approaches to efficiently identify differences in biomolecular patterns between samples [186,187].…”