Benzimidazole, coumrindione and flavone derivatives as alternate UV laser desorption ionization (LDI) matrices for peptides analysis
© Musharraf et al.; licensee Chemistry Central Ltd. 2013
Received: 13 December 2012
Accepted: 17 April 2013
Published: 26 April 2013
Matrix-assisted laser desorption/ionization (MALDI) is a soft ionization mass spectrometric technique, allowing the analysis of bio-molecules and other macromolecules. The matrix molecules require certain characteristic features to serve in the laser desorption/ionization mechanism. Therefore, only a limited number of compounds have been identified as ultraviolet- laser desorption/ionization (UV-LDI) matrices. However, many of these routine matrices generate background signals that useful information is often lost in them. We have reported flavones, coumarindione and benzimidazole derivatives as alternate UV-LDI matrices.
Thirty one compounds have been successfully employed by us as matrices for the analysis of low molecular weight (LMW) peptides (up to 2000 Da). Two peptides, bradykinin and renin substrate tetra-decapeptide were analyzed by using the newly developed matrices. The MS measurements were made after mixing the matrix solution with analyte by using dried droplet sample preparation procedures. The synthesized matrix materials showed better S/N ratios and minimal background signals for low mass range. Furthermore, pico molar concentrations of [Glu1]-fibrinopeptide B human could be easily analyzed with these matrices. Finally, BSA-digest was analyzed and identified through database search against Swiss-Prot by using Mascot.
These results validate the good performance of the synthesized UV-laser desorption/ionization (LDI) matrices for the analysis of low molecular weight peptides.
KeywordsMALDI-MS LDI matrix Benzimidazole derivatives Coumarin derivatives Flavones Peptides
MALDI-MS was developed in the late 1980s and since then it has become a powerful tool for the analysis of large biomolecules, such as proteins, peptides, and nucleic acids, etc. . Two important factors that involved in the ionization of analytes in the MALDI processing are laser and matrices. MALDI techniques commonly employ the use of UV lasers such as nitrogen lasers (337 nm) and frequency-tripled and quadrupled Nd: YAG lasers (355 nm and 266 nm, respectively) as a energy source. Moreover, radiation from IR laser such as Er: YAG lasers and CO2 are also employed . Several modifications have been made on UV lasers to improve the performance. Recently, a modified laser named “smartbeam” has been introduced by the Bruker which is a combination of the speed of a solid-state-laser and the wide range of nitrogen lasers and provides substantial improvement in MALDI performance .
MALDI matrices are generally small organic compounds and act as energy mediators, effectively transferring the laser energy from a source to the surrounding sample molecules, resulting in minimum fragmentation. The method involves both laser ablation and ionization of the matrix/analyte mixture after electronic excitation of the matrix . It is generally observed that effective LDI matrices have an aromatic conjugated system in their structures which enhance the energy absorbing capacity from UV laser. This phenomenon is still relatively unclear therefore certain molecule can act as matrices and others not . Moreover, routinely used commercially available matrices such as HCCA (α-cyano-4-hydroxycinnamic acid), DHB (2,5-dihydroxybenzoic acid) and SA (sinapinic acid) also get ionized, producing large number of background signals in the low-mass region which restricted the use of MALDI for the determination of low molecular weight analyte. In the investigations of low molecular weight compounds, a number of new matrices and many modifications of the MALDI targets have been accomplished [6–25].
In an attempt to correlate the performance of a matrix in a MALDI experiment with its chemical structure, we studied thirty one synthetic alternate matrices for their desorption and ionization of intact small peptides. A large number of derivatives of flavones, coumarindione (coumarin derivative) and benzimidazol with necessary functionalities have been screened as potential matrices without any tedious chemical modification or applying expensive procedures.
Chemicals and reagents
HPLC grade methanol, trifluoro acetic acid, acetone, and acetonitrile were purchased from Sigma-Aldrich (USA). All standards, ([Glu1]-fibrinopeptide B human (Glufib, ≥90%), bradykinin (≥97%) and renin substrate tetra-decapeptide (≥97%), were obtained from Sigma-Aldrich (USA). Cholic acid (≥97%) was obtained from Wako (Japan) while bovine serum albumin (BSA)-digest was purchased from Bruker Daltonics (Germany). Deionized water (Milli-Q) was used throughout the study (Millipore, USA). 2-Phenylenediamine, 4-hydroxy coumarin, ethyl benzoyl acetate, phloroglucinol and the other reagents and solvents used in the preparation of matrices were of synthetic grades, obtained from Sigma-Aldrich (USA).
Preparation of matrix materials
Benzimidazole derivatives were synthesized by reacting together commercially available 2-phenylenediamine and different substituted aromatic aldehydes in N,N-dimethylformamide (DMF). The mixture was heated to reflux for 2 h; the progress of the reaction was monitored by TLC. After completion of the reaction, the mixture was allowed to cool to room temperature. Addition of water resulted in precipitation of a solid material, after filtration, the solid benzimidazole derivatives were obtained in high yields . Coumarindione (coumarin derivative) were synthesized by taking a mixture of different substituted benzaldehyde and 4-hydroxy coumarin in water. Heating and stirring was continued for 1–2 h at 100°C. The reaction progress was monitored by TLC. On completion, the solid was filtered and washed subsequently with boiling water and hexane. After drying in vacuum, the coumarindione was obtained as a solid product. Flavones were synthesized by mixing different substituted aromatic β-ketoesters and substituted phenols, followed by irradiation with microwaves 300 W at 100°C for 3 min. The crude product was dissolved in 10% aq. NaOH and washed with diethyl ether. The product was precipitated by adding concentrated HCl, which was filtered, washed with water and vacuum-dried to obtain the desired flavones .
Preparation of standard solutions
Peptide standards, bradykinin and renin substrate tetra-decapeptide, were dissolved in 0.1% TFA: ACN (1:1), while cholic acid was dissolved in methanol (1 mg/mL). The stock solution of peptide standards was prepared in a 1 mM concentration, and working standard solutions of 25 pM were prepared through the dilution of stock solution. BSA-digest was dissolved in 0.1% trifluoroacetic acid (TFA) in a concentration of 4 pM. [Glu1]-fibrinopeptide B human was used for sensitivity measurements in different concentrations of 1000, 100, 50, 25, 12.5 and 10 pM.
UV–Vis absorption measurements
Synthesized materials of benzimidazole, flavones and coumarindione derivatives were dissolved in methanol having concentration of 1.0-0.5 mM. The absorption maxima was recorded by UV/Visible scanning in the region of 200–800 nm against the reagent blank on a UV/Visible double beam spectrophotometer (Thermo Scientific Evolution 300, UK), є and Log є were calculated at λmax by Lambert-Beer law.
MALDI-MS measurements were carried out on Ultraflex III TOF/TOF (Bruker Daltonics, Bremen, Germany) mass spectrometer, equipped with a smartbeam laser (Nd: YAG, 355 nm, maximum pulse energy of 300 μJ, beam diameter 0.4 mm) and an electrostatic reflector. The focal length of lense is 50 mm. Mass spectra were recorded in reflector mode with the ion source 1 (ISI) set to 25.00 kV, source 2 (ISI) set to 21.50 kV, a lens voltage of 9.51 kV and performed with the laser energies of 55 ± 5%. The validation of data including the baseline subtraction was obtained through Flex analysis. External calibration was carried by using peptide calibration standards (Bruker Daltonics, Bremen,Germany). 0.1% TFA solution was prepared in deionized water and synthetic matrix materials were dissolved in different concentration (1–2 mM) in the solvent system of 0.1% TFA: ACN (1:1). The solution was sonicated for 5 minutes on a sonicator (Ultrasonic LC 38H), followed by centrifugation (Centrifuge 5804R Eppendorf). The supernatant was employed as a matrix. All measurements were made after mixing the matrix solution with analyte at a volume ratio of 1:2, spotted 1.5 μL droplet of mixture on the MALDI target plate (MTP 384, spot diameter 3.5 mm) and allowing the droplet to dry at ambient conditions. The dried droplet preparation method was used for the sample preparation. The dried co-crystallized sample was then analyzed by MALDI-MS. To improve the signal-to-noise ratio, spectral data from every 200 laser shots were summed. Then, the results from 3 different locations on a sample spot were summed. Hence, each point in such spectra corresponds to the summation of over 600 shots. The background spectra of matrices was recorded on off mode (provided in the Additional file 1: Figure S1) while with the analyte, the spectra were recorded on deflection mode in order to minimize all background signals generating from matrix.
Recorded BSA digest spectra with various matrices was submitted to the MASCOT search engine (Matrix Science, London, UK), using UniProt/Swiss-Prot (release July 2010, Homo sapiens, 18055 sequences) as the reference database. Mascot search parameters were as follows: enzyme specificity trypsin, fixed modifications cysteine carbamidomethylation, variable modification methionine oxidation. The maximum number of missed cleavages was set to 3 and mass tolerance at (0.1-0.9 Da). Stereomicroscopic image of matrices spots were recorded by using Nikon SMZ 800 (Japan) fitted with (DS-Vi1) camera.
Results and discussion
The λ max and molar absorptivities of different classes of compounds
Class (I) Benzimidazole derivatives
N, N-Dimethyl-4-(1-phenyl-1H-benzimidazol-2-yl) aniline
Class-11 (Coumarin derivative)
3-[(2,3,4-Trihydroxyphenyl) methyl idene]-2H-chromene-2,4-dione
Signal intensity and S/N ratio of synthetic matrix material with low molecular weight analytes
Renin substrate tetra-decapeptide a
Screening of compounds
All synthetic compounds were prepared for screening in 0.1% TFA: ACN with matrix to analyte volume ratio of 1:2 for the detection of low molecular weight peptides, bradykinin and renin substrate tetra-decapeptide (<2000 Da).
Fourteen compounds of class I (benzimidazole derivatives) possessing λmax around 213–325 nm, were screened. All these new synthetic matrices of class I was comparable to existing MALDI matrix, HCCA in terms of intensity for the detection of peptides. However, the new matrices showed almost no background signals in the low mass range and hence were found to be suitable for the analysis of small peptides. Compounds 5, 8, 9, 11, 12 and 14 showed very high intensity (>1× 105) for peptides (Table 2).
Twelve compounds of class II (coumarindione) were also screened as LDI matrix. All of them showed high intensity (>1× 104) for low molecular weight peptides. Moreover, coumarin derivatives possess λmax between 212–324 nm, most of the compounds showed best results. However, coumarin dyes  and 3-hydroxycoumarin  have been reported as a MALDI matrix for the analysis of proteins and oligodeoxynucleotides, respectively. Five compounds of class III (flavones), possessing λmax between 261–334 nm, were also tested as LDI matrices. Both peptides showed good signal intensity (>1× 104) as presented in Table 2.
Data-base search results of BSA-digest, analyzed with various matrices
Average Mascot Score
Average number of Peptides
% Sequence coverage
Thirty one new matrices for laser desorption/ionization mass spectrometry (LDI-MS) have been studied and offered high performance and sensitivity which make them efficient platform for the analysis of small peptides. A number of conjugated aromatic systems in their structures ensure the laser energy transformations to the analytes. Furthermore, screening of a large number of organic compounds with various functionalities will be helpful for the development of structure–property relationship models, which in turn, could lead to a better understanding of the MALDI mechanism and discovery of better matrices in future.
The authors are thankful to Prof. Dr. Nurul Kabir for providing his lab facility to record the microscopic images.
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