Sample preparation (mass spectrometry)

The sample preparation for mass spectrometry is used for the optimisation of a sample for the analysis in a mass spectrometer (MS).These have depending on their ion source different requirements for volume, concentration, and composition of the analyte solution. Furthermore, in protein mass spectrometry usually the protein of interest is cleaved into peptides before analysis, either by in-gel digestion or by proteolysis in solution.

MALDI

For the MALDI mass spectrometry the sample is usually mixed with a matrix solution and spottet on the target plate. The matrix crystallises together with the sample and the analyte molecules are transferred to the gas phase by the pulsed laser irradiation.

The concentration of salt in the sample is thereby not so critical as it is for elektrospray ionisation. However, interfering signals are observed due to side reactions of the matrix with alkali metal ions which can impair the analysis of the spectra. Nevertheless, a separate desalting step is usually not necessary as in previous steps of sample preparation e.g. in-gel digestion of proteins, the selection of appropriate buffer salts prevents the occurrence of this problem. Furthermore the crystallised matrix/analyte mixture allows removing of salts by washing on the target and the addition of ammonium phosphate before crystallisation can improve the signal quality. " [http://pubs.acs.org/cgi-bin/abstract.cgi/ancham/2004/76/i10/abs/ac035331j.html Smirnov et al, Anal. Chem., 76 (10), S. 2958-2965, 2004] "]

= ESI =

The electrospray ionisation because of its principle of ionisation is more demanding in the characteristics of the sample. In off-line measurements (in contrast to on-line measurements using LC-MS), the analyte solution is applied by a spray capillary to the mass spectrometer. This capillary allows for the application of volumes in the range of tenth of mikroliters, however, the detection limit of the instrument requires in protein analysis a relatively high concentration of the analyte. Therefore, usually a step to increase the concentration of the sample is needed.The evaporation of the solvent in the spray during the ionisation process accounts for the demand of a salt-free analyte solution. Otherwise, with the continuous reduction of the volume of the droplets in the spray the salts would start to aggregate which inhibits the formation of useful analyte ions for the measurement.

A major application for ESI is the field of protein mass spectrometry. Here, the MS is used for the identification and sizing of proteins. The identification of a protein sample can be done in an ESI-MS by de novo sequencing (using tandem mass spectrometry) or peptide mass fingerprinting. Both methods require the previous digestion of proteins to peptides, mostly accomplished enzymatically using proteases. As well for the digestion in solution as for the in-gel digestion buffered solutions are needed, whose content in salts is to high and in analyte is to low for a successful ESI-MS measurement. Therefore, a combined desalting and concentration step is performed. Usually a reversed phase liquid chromatography is used, in which the peptides stay bound to the chromatography matrix whereas the salts are removed by washing. The peptides can be eluted from the matrix by the use of a small volume of a solution containing a large portion of organic solvent, which results in the reduction of the final volume of the analyte. In LC-MS the desalting/concentration is realised with a pre-column, in off-line measurements reversed phase micro columns are used, which can be used directly with microliter pipettes. Here, the peptides are eluted with the spray solution containing an appropriate portion of organic solvent. The resulting solution (usually a few microliters)is enriched with the analyte and, after transfer to the spray capillary, can be directly used in the MS.

See also

*In-gel digestion
*protein mass spectrometry
*tandem mass spectrometry
*Electrospray ionization (ESI)
*MALDI

References