A novel approach using MALDI-TOF/TOF mass spectrometry and prestructured sample supports (AnchorChip Technology) for proteomic profiling and protein identification.

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.

Design of tetrapeptide ligands as inhibitors of the Src SH2 domain.

Src homology-2 (SH2) domains are noncatalytic motifs containing approximately 100 amino acid residues that are involved in intracellular signal transduction. The phosphotyrosine-containing tetrapeptide pTyr-Glu-Glu-Ile (pYEEI) binds to Src SH2 domain with high affinity (K(d)=100 nM). The development of five classes of tetrapeptides as inhibitors for the Src SH2 domain is described. Peptides were prepared via solid-phase peptide synthesis and tested for affinity to Src SH2 domain using a fluorescence polarization based assay. All of the N-terminal substituted pYEEI derivatives (class II) presented binding affinity (IC(50)=of 2.7-8.6 microM) comparable to pYEEI (IC(50)=6.5 microM) in this assay. C-Terminal substituted pYEEI derivatives (class III) showed a lower binding affinity with IC(50) values of 34-41 microM. Amino-substituted phenylalanine derivatives (class IV) showed weak binding affinities (IC(50)=16-153 microM). Other substitutions on phenyl ring (class I) or the replacement of the phenyl ring with other cyclic groups (class V) dramatically decreased the binding of tetrapeptides to Src SH2 (IC(50)>100 microM). The ability of pYEEI and several of the tetrapeptides to inhibit the growth of cancer cells were assessed in a cell-based proliferation assay in human embryonic kidney (HEK) 293 tumor cells. The binding affinity of several of tested compounds against Src SH2 domain correlates with antiproliferative activity in 293T cells. None of the compounds showed any significant antifungal activity against Candida albicans ATCC 14053 at the maximum tested concentration of 10 microM. Overall, these results provided the structure-activity relationships for some FEEI and YEEI derivatives designed as Src SH2 domain inhibitors.