Sample preparation (mass spectrometry)
The mass spectrometric sample preparation is used to optimize a sample for analysis in a mass spectrometer . Depending on the ionization process, these have different requirements in terms of volume, concentration and composition of the analyte solution.
MALDI
For MALDI mass spectrometry, the sample is traditionally mixed with a matrix solution and applied to the target. There the matrix crystallizes out together with the sample and the analyte molecules are transferred into the gas phase by the pulsed laser radiation. The salt content of the sample is not as critical as, for example, with electrospray ionization (= ESI), but interfering signals can be caused by side reactions of the matrix with e.g. B. Alkali metal ions come, which affect the evaluability of the spectra. A separate desalination step is usually not necessary, since the appropriate selection of the buffer salts in the preceding steps of sample preparation z. B. the in-gel digestion of proteins for protein identification by means of peptide mass fingerprint , the use of alkali metal salts can be avoided. Furthermore, washing the crystallized matrix / analyte mixture on the target or adding ammonium phosphate to the matrix / analyte solution can improve the signal quality.
IT I
The electrospray ionization is due to the Ionisationsprinzips higher demands on the properties of the sample. The volume of the needle, which is used for offline measurements (ie the sample is not introduced into the mass spectrometer via the coupling to liquid chromatography ), prevents larger sample volumes. The detection limit, which is higher than that of MALDI , usually requires a concentration of the sample. The evaporation of the solvent during the spray places high demands on the salt-free sample solution, since otherwise the salts begin to crystallize with the continuous reduction of the volume in the spray or needle and thus make a successful measurement impossible. An important application of ESI-MS devices is in the field of proteomics . The mass spectrometer is used for the identification and size determination of proteins. The identification of the protein samples can be done with ESI measurements via de novo peptide sequencing or peptide mass fingerprinting . Both methods require a previous digestion of the protein into peptides , which is usually done enzymatically with the help of proteases . Both for digestion in solution and for in-gel digestion , buffered solutions are required whose salt content and volume are too high and whose analyte content is too low for the ESI measurement. Therefore a combined desalination and concentration step is carried out before the measurement. Typically, this is a reverse phase liquid chromatography (RP-LC) is used, wherein the peptides bound to the chromatography matrix, however, the salts can be removed by washing. The bound peptides can be eluted with a small volume of organic solvent. With the LC / MS coupling , the desalination / concentration is mostly realized with a guard column, for off-line measurements RP micro columns are used which can be attached directly to microliter pipettes . The elution takes place here with the spray solution, which contains a correspondingly high proportion of organic solvents. The spray solution provided with the enriched analyte can then be transferred directly into the spray capillary and thus into the mass spectrometer.
Individual evidence
- ↑ IP Smirnov, X. Zhu, T. Taylor, Y. Huang, P. Ross, IA Papayanopoulos, SA Martin, DJ Pappin: Suppression of α-Cyano-4-hydroxycinnamic Acid Matrix Clusters and Reduction of Chemical Noise in MALDI-TOF Mass Spectrometry , in: Anal. Chem. 2004 , 76 , 2958-2965; doi : 10.1021 / ac035331j .