4-Chloro-α-cyanocinnamic acid is an advanced, rationally designed MALDI matrix

Abstract

Matrix-assisted laser desorption ionization (MALDI) has become an enabling technology for the fields of protein mass spectrometry (MS) and proteomics. Despite its widespread use, for example, in protein identification via peptide mass fingerprinting, a comprehensive model for the generation of free gas-phase ions has not yet been developed. All matrices in use today, such as ␣-cyano-4-hydroxycinnamic acid (CHCA), have been found empirically and stem from the early days of MALDI. By systematic and targeted variation of the functional groups of the ␣-cyanocinnamic acid core unit, 4-chloro-␣-cyanocinnamic acid (Cl-CCA) was selected and synthesized, and it exhibited outstanding matrix properties. Key features are a substantial increase in sensitivity and a considerably enhanced peptide recovery in proteomic analyses because of a much more uniform response to peptides of different basicity. Using Cl-CCA as a matrix for a 1 fmol bovine serum albumin (BSA) in-solution digest, the sequence coverage is raised to 48%, compared with 4% for CHCA. For a gel band containing 25 fmol of BSA, unambiguous protein identification becomes possible with Cl-CCA. These findings also imply ion formation via a chemical ionization mechanism with proton transfer from a reactive protonated matrix species to the peptide analytes. The considerable increase in performance promises to have a strong impact on future analytical applications of MALDI, because current sensitivity limits are overcome and more comprehensive analyses come into reach.ionization mechanism ͉ proton transfer M atrix-assisted laser desorption ionization (MALDI) (1-3) relies on a cocrystallization of the analytes of interest with an excess of small organic compounds, the matrix, which is accomplished by either simply drying Ϸ 1-l droplet of a solution containing matrix and analyte (dried-droplet preparation) (2, 3) or by depositing a droplet of the analyte solution onto a prespotted matrix layer (surface preparation) (4). The matrix exhibits a strong absorption at the laser wavelength used (typically 337 nm or 355 nm in the UV), which, after irradiation with a short pulse of laser light, leads to the ablation of a shallow surface region into the vacuum of the mass spectrometer, the release of intact gaseous matrix and analyte molecules, and their partial ionization. Since its introduction in the 1980s, MALDI has found widespread use in the mass spectrometric analysis of biological macromolecules but recently also has been used increasingly for small-molecule analysis (5, 6).However, the main field of application of MALDI mass spectrometry (MS) today is protein identification in proteomic schemes by using the peptide mass fingerprint (PMF) approach (7), often supplemented by MALDI-time-of-f light/time-offlight (TOF/TOF) analyses. ␣-Cyano-4-hydroxycinnamic acid (CHCA) (8) as a matrix (in positive-ion mode) allows the researcher to identify proteins via MS or MS/MS analysis of their proteolytic peptide products (typically generated by trypsin) and by using protein or DNA seque...

The new matrix 4-Chloro-α-cyanocinnamic acid allows the detection of phosphatidylethanolamine chloramines by MALDI-TOF mass spectrometry

“…For this purpose the PA of the energetically lowest conformer of the dichlorinated ethanolamine head group of Cl 2 DPPE was calculated using the B3LYP method with the 6-311ϩϩG(2d,2p) basis set (SI Figure 4). The PA was calculated to be 789.9 kJ/mol and is, therefore, about 42 kJ/mol lower compared to the experimentally determined PA value of 832 kJ/mol for ClCCA [10]. As a result, no protonation of Cl 2 DPPE can occur which explains the missing of the corresponding Cl 2 DPPE ϩ H ϩ signals.…”

“…Due to the low proton affinity (PA) of ClCCA, this compound was already successfully used for the analysis of protonable analytes like proteins and peptides and provided spectra of superior quality in comparison to common MALDI matrices [10], but this new matrix was not yet used for the detection of chloramines. It was supposed that because of the clear detection of sodium cation adducts of the DPPE dichloramine not only the PA of ClCCA but also the sodium cation affinity (SCA) is lowered compared with common MALDI matrices.…”

“…We already reported that in comparison with established matrices, ClCCA possesses the lowest matrix proton affinity (PA) known so far, which increases the protonation efficiency of analytes, as, e.g., peptides, phosphopeptides, or proteins [10]. Additionally, we calculated the sodium cation affinity (SCA) of this matrix and further discuss differences of the matrices on the basis of studies regarding their effective plume temperatures and initial ion velocities.…”

High-Throughput IgG Fc N-Glycosylation Profiling by Mass Spectrometry of Glycopeptides

“…Subsequently, 1 μL of 5 mg/mL 4-chloro-α-cyanocinnamic acid (Cl-CCA; 95% purity; Bionet Research, Camelford, Cornwall, U.K.) in 50% acetonitrile was applied on top of each sample and allowed to dry. 24,25 Glycopeptides were analyzed on an UltrafleX II MALDI-TOF/TOF mass spectrometer (Bruker Daltonics), which was operated in the negative-ion reflectron mode. Ions between m/z 1000 and 3800 were recorded.…”

Acid–base-driven matrix-assisted mass spectrometry for targeted metabolomics

“…Such simple matrix attributes can be calculated by using DFT methods, which opens the door for rational in silico MAILD matrix design. Our concept represents another level in rational design of matrices initiated by Karas and coworkers (25).…”