A dual fluorescent/MALDI chip platform for analyzing enzymatic activity and for protein profiling

Abstract

Being able to rapidly and sensitively detect specific enzymatic products is important when screening biological samples for enzymatic activity. We present a simple method for assaying protease activity in the presence of protease inhibitors (PIs) by measuring tryptic peptide accumulation on copolymer pMALDI target chips using a dual fluorescence/MALDI-TOF-MS read-out. The small platform of the chip accommodates microliter amounts of sample and allows for rapid protein digestion. Fluorescamine labeling of tryptic peptides is used to indicate the proteolytic activity and is shown to be an affordable, simple process, yielding a strong fluorescence signal with a low background. Subsequent MALDI-TOF-MS analysis, performed in the same sample well, or in a parallel well without adding fluorescamine, detects the specific tryptic peptides and provides confidence in the assay. The dual read-out method was applied to screen the inhibition activity of plant PIs, components of plant defense against herbivores and pathogens. Extracts of PIs from Solanum nigrum and trypsin were applied together to a pMALDI chip on which a suitable substrate was adsorbed. The fluorescence and MALDI-TOF-MS signal decrease were associated with the inhibitory effect of the PIs on trypsin. The developed platform can be modified to screen novel protease inhibitors, namely, those potentially useful for treating or preventing infection by viruses, including HIV and hepatitis C.

Enzymatic Deposition of Silver Particles for Detecting Protease Activity

“…This array‐based assay can be used to detect multiple proteases with different oligopeptides simultaneously. However, the LOD of this LC‐based protease assay is high (0.05 μg mL −1 ) compared to other array‐based protease assays such as matrix‐assisted laser desorption/ionization–time of flight (MALDI‐TOF) as mentioned above.…”

Lab‐on‐a‐plate: Extending the functionality of MALDI‐MS and LDI‐MS targets

“…Fluorometric detection is non-destructive and will therefore not affect the subsequent ionization step. Examples include single cell visualization with fluorescence followed by NIMS (Northen et al, 2007), a dual fluorescent/MALDI chip platform for analyzing enzymatic activity (Halim et al, 2009), gel-pad microarrays formed by patterning porous silicon for dual-mode detection of proteins by fluorescence and MALDI-MS (Chen et al, 2009), and a novel strategy for following chemical reactions carried out on the zeptomol (10 À21 mol) scale using ''attoreactors'' that are analyzed by both fluorescence and MALDI-MS (Anzenbacher & Palacios, 2009; see also Section IV.C). A single paper has even appeared dealing with DNA detection on a planar microwell plastic chip compatible with optical/fluorescence detection, MALDI-MS, and atomic force microscopic imaging (Ibáñez et al, 2008).…”

A Sol-gel Integrated Dual-readout Microarray Platform for Quantification and Identification of Prostate-specific Antigen

“…9 The concept was first tested by Finnskog et al 8 using antibody arrays on a porous silicon (pSi) surface for detection and identification of IgG and AngII by fluorescent and MALDI-TOF MS readouts of affinity binding and mass. 8 Several types of material can be used for dual-readout platforms; these include pSi 8 , copolymer MALDI array chips, 10 and indium tin oxide (ITO) glycan slides. 11 These materials provide a large surface area and facilitate immobilization of target molecules.…”