4-Chloro-alpha-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 alpha-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 alpha-cyanocinnamic acid core unit, 4-chloro-alpha-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.
Project description:The matrix application technique is critical to the success of a matrix-assisted laser desorption/ionization (MALDI) experiment. This work presents a systematic study aiming to evaluate three different matrix application techniques for MALDI mass spectrometric imaging (MSI) of endogenous metabolites from legume plant, Medicago truncatula, root nodules. Airbrush, automatic sprayer, and sublimation matrix application methods were optimized individually for detection of metabolites in the positive ionization mode exploiting the two most widely used MALDI matrices, 2,5-dihydroxybenzoic acid (DHB) and α-cyano-4-hydroxycinnamic acid (CHCA). Analytical reproducibility and analyte diffusion were examined and compared side-by-side for each method. When using DHB, the optimized method developed for the automatic matrix sprayer system resulted in approximately double the number of metabolites detected when compared to sublimation and airbrush. The automatic sprayer method also showed more reproducible results and less analyte diffusion than the airbrush method. Sublimation matrix deposition yielded high spatial resolution and reproducibility but fewer analytes in the higher m/z range (500-1000 m/z). When the samples were placed in a humidity chamber after sublimation, there was enhanced detection of higher mass metabolites but increased analyte diffusion in the lower mass range. When using CHCA, the optimized automatic sprayer method and humidified sublimation method resulted in double the number of metabolites detected compared to standard airbrush method.
Project description:BACKGROUND:Unravelling the serum proteome is the subject of intensified research. In this regard, two-dimensional electrophoresis coupled with MALDI MS analysis is still one of the most commonly used method. Despite some improvements, there is the need for better protocols to enable comprehensive identification of serum proteins.Here we report a combination of two proteomic strategies, zoom in acidic and neutral part of 2-D gels and an application of two optimised matrix preparations for MALDI-MS analyses to simplify serum proteome mapping. RESULTS:Mouse serum proteins were separated by 2-D electrophoresis at the pH ranges 3-10 and 4-7, respectively. Then in gel tryptic digests were analysed by MALDI-MS. Notably, sample-matrix preparations consisted of either a thin-layer alpha-ciano-4-hydroxycinnamic acid (CHCA) matrix deposition or a matrix-layer 2,5-dihydroxybenzoic acid (DHB). This enabled an identification of 90 proteins. The herein reported method enhanced identification of proteins by 32% when compared with previously published studies of mouse serum proteins, using the same approaches. Furthermore, experimental improvements of matrix preparations enabled automatic identification of mouse proteins, even when one of the two matrices failed. CONCLUSION:We report a simple and reliable protocol for serum proteome analysis that combines an optimized resolution of 2-D gels spots and improved sample-matrix preparations for MALDI-MS analysis. The protocol allowed automated data acquisition for both CHCA and DHB and simplified the MS data acquisition therefore avoiding time-consuming procedures. The simplicity and reliability of the developed protocol may be applied universally.
Project description:Laser Desorption Ionization Mass Spectrometry employs matrix which is co-crystallised with the analyte to achieve "soft ionization" that is the formation of ions without fragmentation. A variety of matrix-free and matrix-assisted LDI techniques and matrices have been reported to date. LDI has been achieved using ultra fine metal powders (UFMPs), desorption ionisation on silicon (DIOS), sol-gel assisted laser desorption/ionization (SGALDI), as well as with common MALDI matrices such as 2,5-dihydroxy benzoic acid (DHB), 3,5-dimethoxy-4-hydroxycinnamic acid (SA), alpha-cyano-4-hydroxycinnamic acid (CHCA) to name a few. A variety of matrix additives have been shown to improve matrix assisted desorption, including silicon nanowires (SiNW), carbon nanotubes (CNT), metal nanoparticles and nanodots. To our knowledge no evidence exists for the application of highly fluorescent CdSe/ZnS quantum dots to enhance MALDI desorption of biological samples. Here we report that although CdSe/ZnS quantum dots on their own can not substitute matrix in MALDI-MS, their presence has a moderately positive effect on MALDI desorption, improves the signal-to-noise ratio, peak quality and increases the number of detected peptides and the overall sequence coverage.
Project description:MALDI MS imaging (MSI) is a powerful analytical tool for spatial peptide detection in heterogeneous tissues. Proper sample preparation is crucial to achieve high quality, reproducible measurements. Here we developed an optimized protocol for spatially resolved proteolytic peptide detection with MALDI time-of-flight MSI of fresh frozen prostate tissue sections. The parameters tested included four different tissue washes, four methods of protein denaturation, four methods of trypsin digestion (different trypsin densities, sprayers, and incubation times), and five matrix deposition methods (different sprayers, settings, and matrix concentrations). Evaluation criteria were the number of detected and excluded peaks, percentage of high mass peaks, signal-to-noise ratio, spatial localization, and average intensities of identified peptides, all of which were integrated into a weighted quality evaluation scoring system. Based on these scores, the optimized protocol included an ice-cold EtOH+H<sub>2</sub> O wash, a 5 min heating step at 95°C, tryptic digestion incubated for 17h at 37°C and CHCA matrix deposited at a final amount of 1.8 μg/mm<sup>2</sup> . Including a heat-induced protein denaturation step after tissue wash is a new methodological approach that could be useful also for other tissue types. This optimized protocol for spatial peptide detection using MALDI MSI facilitates future biomarker discovery in prostate cancer and may be useful in studies of other tissue types.
Project description:Peptides larger than 3-4?kDa, such as neuropeptide Y (NPY), orexin-B, and alpha-MSH, have practical issues that arise when conducting direct and sensitive quantitative liquid chromatography (LC) orbitrap-FT mass spectrometry (MS) due to their adsorption and low ionization efficiency, especially in standard solutions. A mixing solvent consisting of 0.5% trifluoroacetic acid (TFA) and 35-50% aq. acetonitrile was developed as the standard NPY for creating calibration curves, as well as a matrix to block the experimental tube surface to minimize adsorption. The mixture matrix effectively blocked non-specific adsorption of the standard peptides with tryptic digested bovine serum albumin (BSA) (small fragment peptides) and orexin-B (a large chain peptide). A sample containing 1?:?100 peptide:water was detected in the developed sample solution. Finally, 2 to 1,000?fmol/?L NPY could be analyzed quantitatively and reproducibly using conventional LC-MS. Parameters of the calibration curves, such as X-intercept, Bias (%), and relative standard deviation (RSD), were adjusted to optimize the sample solutions and the sensitive and quantitative LC-MS analyses.
Project description:Croconaine dyes are appealing molecules synthesized via the condensation of croconic acid and reactive electron-donating aromatic or heterocyclic systems. Here, matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) investigation of indolenine-based croconaines is presented for the first time. Archetype proton-transfer matrices, such as 2,5-dihydroxybenzoic acid (DHB) and ?-cyano-4-hydroxycinnamic acid (CHCA), 9-aminoacridine (9AA) as the protonating/deprotonating matrix, and electron-transfer (ET) secondary-reaction matrices, such as 1,5-diaminonapthalene (DAN) and trans-2-[3-(4-t-butyl-phenyl)-2-methyl-2-propenylidene]malononitrile (DCTB), were investigated. DHB, CHCA, and 9AA generate a mix of odd-electron molecular ions and protonated, sodiated, and potassiated adducts. Among the ET matrices, DAN was found to be capable of directing the ionization process toward the exclusive formation of odd-electron molecular ions M+• without fragmentation. MALDI tandem MS provides useful structural characterization of croconaine dyes, thus making identification very straightforward for all investigated compounds. Interestingly the fragmentation of bromo-containing croconaines revealed, for the first time, the gas-phase formation of a bromime cation [Br]+.
Project description:An electrospray-assisted laser desorption/ionization source with an infrared OPO laser (IR-ELDI) was constructed and optimized for peptide and protein mass spectrometry analysis. Similar to ELDI with an ultraviolet laser, IR-ELDI generates multiply charged molecules for peptides and proteins measured under ambient sampling conditions. Both samples in the dried state and analyte solutions can be directly measured by IR-ELDI without the presence of a conventional MALDI matrix. However, the analysis of sample solutions is shown to greatly enhance the sensitivity of the mass spectrometry measurement, as a 100-fold sensitivity gain for peptide measurements was measured. The limit of detection of IR-ELDI was determined to be 250 fmol for bradykinin (1.1 kDa), 100 fmol for ubiquitin (8.6 kDa), and 500 fmol for carbonic anhydrase (29 kDa). IR-ELDI is amenable for MS and MSn analysis for proteins up to 80 kDa transferrin. IR-ELDI-MS may be a useful tool for protein sequencing analysis from complex biological matrices, with minimal sample preparation required.
Project description:Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has gained popularity in recent years for rapid bacterial identification, mostly at the genus or species level. In this study, a rapid method to identify the Escherichia coli flagellar antigen (H antigen) at the subspecies level was developed using a MALDI-TOF MS platform with high specificity and sensitivity. Flagella were trapped on a filter membrane, and on-filter trypsin digestion was performed. The tryptic digests of each flagellin then were collected and analyzed by MALDI-TOF MS through peptide mass fingerprinting. Sixty-one reference strains containing all 53 H types and 85 clinical strains were tested and compared to serotyping designations. Whole-genome sequencing was used to resolve conflicting results between the two methods. It was found that DHB (2,5-dihydroxybenzoic acid) worked better than CHCA (?-cyano-4-hydroxycinnamic acid) as the matrix for MALDI-TOF MS, with higher confidence during protein identification. After method optimization, reference strains representing all 53 E. coli H types were identified correctly by MALDI-TOF MS. A custom E. coli flagellar/H antigen database was crucial for clearly identifying the E. coli H antigens. Of 85 clinical isolates tested by MALDI-TOF MS-H, 75 identified MS-H types (88.2%) matched results obtained from traditional serotyping. Among 10 isolates where the results of MALDI-TOF MS-H and serotyping did not agree, 60% of H types characterized by whole-genome sequencing agreed with those identified by MALDI-TOF MS-H, compared to only 20% by serotyping. This MALDI-TOF MS-H platform can be used for rapid and cost-effective E. coli H antigen identification, especially during E. coli outbreaks.
Project description:Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) using conventional organic matrices for detection of small molecules has some limitations, such as heterogeneous analyte/matrix co-crystals, as well as interference of matrices in the low-molecular-weight range. In this work, a zeolite framework nanomaterial, TS-1, was applied as a MALDI matrix for the analysis of small molecules by MALDI-MS for the first time. To improve the signal intensity and reproducibility, TS-1 was modified with polydopamine (TS-1@PDA). Using TS-1@PDA as a matrix, organic substances in the low-molecular-weight region such as amino acids, nucleosides, peptides, oligosaccharides, and fatty acids can be detected by MALDI-MS in positive ion mode. Compared with traditional organic matrices like 2,5-dihydroxybenzoic acid (2,5-DHB) and ?-cyano-4-hydroxycinnamic acid (CHCA), TS-1@PDA has the advantages including the formation of uniform sample spots, small background interference at low molecular weight, and better salt tolerance. Furthermore, this matrix was employed for the analysis of endogenous glucose in urine samples, and the level of glucose was quantified with a linear range of 0-10 mM (R 2 > 0.98). The results demonstrated that TS-1@PDA has the potential to be used as an effective MALDI matrix for the analysis of small molecules in biological samples with excellent reproducibility and moderate sensitivity.
Project description:Matrix-assisted laser desorption/ionization (MALDI) mass spectrometric imaging (MSI) has been employed as a detection method for both capillary electrophoresis (CE)-MALDI and liquid chromatography (LC)-MALDI analyses. Based on our previous studies, here we report a new interface to couple LC with MSI by employing an automated matrix sprayer. The LC trace is directly collected on a ground stainless steel MALDI plate and dried. The matrix is sprayed on the MALDI plate using a programmable matrix sprayer. With the highly uniform matrix layers produced from the sprayer, the MS image signal quality is significantly improved with enhanced signal-to-noise ratios for analyte peaks. With the programmable matrix application and imaging MS data acquisition, the new LC-MSI platform exhibits highly stable and reproducible performance. A total of 87 bovine serum albumin (BSA) tryptic peptides and 295 putative neuropeptides from blue crab pericardial organs have been observed with LC-MSI analysis, exhibiting better performance in terms of peptide coverage than regular LC-MALDI with discrete spot collection and our previously reported LC-MSI interface with the matrix being delivered by a capillary. In addition to relative quantitation with isotopic labeling as we have previously demonstrated, we performed the first absolute quantitation using the new LC-MSI platform and obtained accurate quantitation results for neuropeptides, indicating great potential for quantitative analysis of complex samples.