Recognition of cysteine-containing peptides through prompt fragmentation of the 4-dimethylaminophenylazophenyl-4'-maleimide derivative during analysis by MALDI-MS

Abstract

Under specified UV-MALDI conditions, the 4-dimethylaminophenylazophenyl-4'-maleimide (DABMI) derivative of cysteine-containing peptides undergoes prompt fragmentation to produce a characteristic mass spectral pattern. As reported previously by others, derivatization with chromophoric DABMI allows cysteine-containing peptides and proteins to be tracked during HPLC by absorbance at upper UV and visible wavelengths. Here, we describe methodology by which these same peptide derivatives can be recognized by their distinctive MALDI mass spectral fragmentation pattern, then mass mapped, allowing for easy, rapid identification of cysteine-containing peptides.

Figure 1.

Positive ion MALDI mass spectrum taken in reflector mode of DABMI-IGF with CHCA as the matrix. Structures are proposed for the represented ions; the charge is assumed to be located on the peptide (represented by R).

Figure 2.

RP-HPLC chromatogram of a DABMI-labeled peptic digest of native TH (∼60 pmole total protein). Peptides were eluted with a gradient (shown) of acetonitrile containing 0.1% trifluoroacetic acid and monitored by absorbance at 508 nm. In chromatograms of samples containing no peptides, the three largest peaks (indicated by * here) represent DABMI reagent and DABMI-related degradation products.

Figure 3.

Positive ion linear mode MALDI mass spectrum of a fraction collected between 72.25 and 73.65 min during a chromatographic run analogous to that shown in Fig. 2▶, but with a ∼4.5-fold greater quantity of the sample. Evidence for two DABMI-labeled (cysteine-containing) peptides is highlighted. One MH⁺ ion of a DABMI-labeled peptide is represented by a peak at m/z 1385.0, and the other by a peak at m/z 2004.4. The peaks for the accompanying fragmentation products are indicated with # in the inset spectra.