Investigation of Enzyme Kinetics Using Quench−Flow Techniques with MALDI-TOF Mass Spectrometry

Abstract

Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry is combined off-line with rapid chemical quench-flow methods to investigate the pre-steady-state kinetics of a protein-tyrosine phosphatase (PTPase). PTPase kinetics are generally interrogated spectrophotometrically by the employment of an artificial, chromophoric substrate. However, that methodology places a constraint on the experiment, hampering studies of natural, biochemically relevant substrates that do not incorporate a chromophore. The mass spectrometric assay reported herein is based on the formation of a covalent phosphoenzyme intermediate during substrate turnover. This species is generated in the reaction regardless of the substrate studied and has a molecular weight 80 Da greater than that of the native enzyme. By following the appearance of this intermediate in a time-resolved manner, we can successfully measure pre-steady-state kinetics, regardless of the incorporation of a chromophore. The strengths of the mass-spectrometric assay are its uniform response to all substrates, simple and direct detection of covalent enzyme-substrate intermediates, and facile identification of enzyme heterogeneities that may affect enzymatic activity.

Exosite interactions contribute to tension-induced cleavage of von Willebrand factor by the antithrombotic ADAMTS13 metalloprotease

“…Samples (1 l) were mixed with 250 fmol (1 l) of synthetic peptide standard (Sigma) having leucine at position 3 isotopically labeled with 13 C and 15 N (GP ([ 13 C 15 N]L)GSDREQAPNLVY; mass, 1,622). Aliquots (0.5 l) were mixed with an equal volume of ␣-cyano-4-hydroxycinnamic acid matrix (6.2 mg/ml in methanol/ acetonitrile/water ϭ 36/56/8; Agilent Technologies, Santa Clara, CA) and analyzed by MALDI-TOF MS (33). Linearity of the assay was confirmed by constructing standard curves with a similar product peptide (CDREQAPNLVY; mass, 1,307) and the standard peptide.…”

Monitoring enzyme reaction and screening of inhibitors of acetylcholinesterase by quantitative matrix-assisted laser desorption/ionization fourier transform mass spectrometry

“…In recent years, mass spectrometry (MS)-based assays have attracted great attention for high-throughput screening. MS offers the significant advantage that it does not require analytes to be labeled, either by direct attachment of fluorescent and radioactive labels or by binding of antibodies, and therefore offers greater flexibility in experiments [12][13][14]. In the past decade, considerable effort was invested to develop MS-based detection schemes capable of assaying inhibition of enzyme-mediated reactions [15,16].…”

Determination of enzyme/substrate specificity constants using a multiple substrate ESI-MS assay

“…The development of soft ionization techniques, such as electrospray ionization (ESI) and matrix-assisted laser desorption (MALDI), has made mass spectrometry an excellent complementary technique to conventional spectrophotometric methods for studying enzyme kinetics [23][24][25][26][27][28][29]. With the improvements of resolving power, sensitivity, and versatility, mass spectrometry has become a highly competitive analytical method for detection and characterization of biochemical reaction intermediates that can be used to elucidate enzymatic reaction pathways and catalytic mechanisms [16 -22].…”