1. On-target separation of analyte with 3-aminoquinoline/α-cyano-4-hydroxycinnamic acid liquid matrix for matrix-assisted laser desorption/ionization mass spectrometry
Sadanori Sekiya, Kenichi Taniguchi, Koichi Tanaka Rapid Commun Mass Spectrom. 2012 Mar 30;26(6):693-700. doi: 10.1002/rcm.6148.
3-Aminoquinoline/α-cyano-4-hydroxycinnamic acid (3AQ/CHCA) is a liquid matrix (LM), which was reported by Kumar et al. in 1996 for matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. It is a viscous liquid and has some advantages of durability of ion generation by a self-healing surface and quantitative performance. In this study, we found a novel aspect of 3AQ/CHCA as a MALDI matrix, which converges hydrophilic material into the center of the droplet of analyte-3AQ/CHCA mixture on a MALDI sample target well during the process of evaporation of water derived from analyte solvent. This feature made it possible to separate not only the buffer components, but also the peptides and oligosaccharides from one another within 3AQ/CHCA. The MALDI imaging analyses of the analyte-3AQ/CHCA droplet indicated that the oligosaccharides and the peptides were distributed in the center and in the whole area around the center of 3AQ/CHCA, respectively. This 'on-target separation' effect was also applicable to glycoprotein digests such as ribonuclease B. These features of 3AQ/CHCA liquid matrix eliminate the requirement for pretreatment, and reduce sample handling losses thus resulting in the improvement of throughput and sensitivity.
2. An optimized matrix-assisted laser desorption/ionization sample preparation using a liquid matrix, 3-aminoquinoline/α-cyano-4-hydroxycinnamic acid, for phosphopeptides
Yuko Fukuyama, Kohei Takeyama, Shin-ichirou Kawabata, Shinichi Iwamoto, Koichi Tanaka Rapid Commun Mass Spectrom. 2012 Oct 30;26(20):2454-60. doi: 10.1002/rcm.6363.
Rationale: A liquid matrix, 3-aminoquinoline (3-AQ)/α-cyano-4-hydroxycinnamic acid (CHCA), introduced by Kolli et al. in 1996 for matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), has been reported for peptides and proteins, oligonucleotides, oligosaccharides, and glycopeptides. However, it has not been validated for phosphopeptides. Methods: We optimized sample preparation using 3-AQ/CHCA for phosphopeptides. The sensitivity of six phosphopeptide species as isolated or in digests was systematically evaluated by using MALDI-quadropole ion trap (QIT)-time of flight (TOF) MS in positive and negative ion modes, and compared with the conventional methods using a solid matrix, 2,5-dihydroxybenzoic acid (2,5-DHB). Results: The sensitivity of mono- and tetraphosphopeptides was improved 10- to 10 000-fold with the optimized preparation method using 3-AQ/CHCA compared with the conventional methods using 2,5-DHB. Improvement by 3-AQ/CHCA itself was 10-fold. Adding ammonium dihydrogen phosphate or an analyte solvent composition was also effectively improved the sensitivity. Phosphopeptides in isolated form or in digests were detected at femto- or subfemtomole levels. Conclusions: Sensitivity of phosphopeptides was improved by the optimized sample preparation method using 3-AQ/CHCA compared with the conventional method using 2,5-DHB. The validation of 3-AQ/CHCA for phosphopeptides was systematically confirmed, expanding the potential of this matrix to phosphoproteomics.
3. Comparison between the matrices alpha-cyano-4-hydroxycinnamic acid and 4-chloro-alpha-cyanocinnamic acid for trypsin, chymotrypsin, and pepsin digestions by MALDI-TOF mass spectrometry
Thorsten W Jaskolla, Dimitrios G Papasotiriou, Michael Karas J Proteome Res. 2009 Jul;8(7):3588-97. doi: 10.1021/pr900274s.
The performance of the recently developed 4-chloro-alpha-cyanocinnamic acid (Cl-CCA) matrix-assisted laser desorption ionization mass spectrometry (MALDI MS) matrix was investigated in comparison to the most widely used matrix alpha-cyano-4-hydroxycinnamic acid (CHCA). For this purpose, in-solution digestions of standard proteins in the low femtomole range with the proteases trypsin, chymotrypsin, and pepsin were used as analytes. For all protein-protease combinations, Cl-CCA revealed to be highly superior in terms of number of identified peptides, obtained sequence coverages and peptide detection reproducibility. A deeper inspection of the detected peptide signals with regard to both physicochemical peptide properties (their isoelectric point) and mass spectrometric performance (signal-to-noise ratios and mass accuracies) showed that the progress achieved with Cl-CCA is due to the detection of numerous acidic to neutral peptides. Moreover, the higher Cl-CCA sensitivity allowed for the detection of numerous additional phosphopeptides, all of which were verified by means of MS/MS investigations. The occurrence of strong signals of doubly charged peptides which is exclusively observed for the Cl-CCA matrix can be traced back to the peptide amino-acid composition, that is, the presence of a high number of basic amino acids (Arg, Lys, and His) and is thus more pronounced for nontryptic protein digests. These observed improvements well agree with an increased protonation reactivity of Cl-CCA and are more pronounced with a decreasing level of protease specificity and decreasing sample amounts.