Ratiometric pulsed-alkylation mass spectrometry: Method development and application for the study of protein folding and of chelate stability in metalloproteins

Ratiometric Pulsed-Alkylation Mass Spectrometry (PA/MS) has been developed as a new technique in bioanalytical chemistry, which measures the reactivity of cysteine residues as a site-selective probe of proteins in different states. Pulsed-alkylation involves a pulse of modification of reactive thiolates with N-d₅-ethylmaleimide ( d₅-NEM) at a variable pulsed-time, t, followed by a chase with a 10-fold molar excess of H₅-N-ethylmaleimide (H₅-NEM) to fully derivatized the cysteine thiolates. During the chase, the fully alkylated samples are proteolyzed with suitable protease and the resulting peptides subjected to high-resolution matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Identification of the mass peaks corresponding to d₅-NEM and H₅-NEM derivatized peptides and peak area integration allows quantitation of the molar fractions of d₅-NEM and H₅-NEM differentiated mass peaks as a function of pulse time, t, with the resulting data analyzed with an appropriate kinetic model to extract the apparent constant for the reactivity of a thiolate (k_i app).; The method has been applied to study the structure of a stable unfolding intermediate of native bacterial luciferase, an αβ heterodimer, as well as the conformation of the β₂ homodimer, a kinetically trapped off-pathway folding intermediate. In addition, ratiometric PA/MS has been used to extract the pseudo-first order rate constants for the reactivity of cysteine pairs in individual Cys₂-His₂ zinc fingers of human MTF-1, a zinc metalloregulatory protein involved in zinc homeostasis, and in S. aureus CadC, a Cd/Pb/Bi heavy metal sensor protein. These results provide new functional insights into metal chelate stability and in coordination chemistry in distinct metalloregulatory metal complexes.; Ratiometric PA/MS has been demonstrated to be a rapid and sensitive technique to implement and relatively adaptable to a wide variety of solution conditions required by different protein systems. More importantly, it has the potential to gain wide-ranging application to the study of enzyme catalysis, pertinent to those that employ the cysteine thiolate nucleophile in their reaction chemistry, protein-protein, and protein-DNA interactions, in addition to the area of protein folding and coordination of metal ions.