Chemical Sciences: A Manual for CSIR-UGC National Eligibility Test for Lectureship and JRF/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.

Matrix-assisted laser desorption/ionizationEdit

For matrix-assisted laser desorption/ionization (MALDI) mass spectrometry the sample is usually mixed with a matrix solution and spotted on the target plate.[1] 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 electrospray ionization. However, interfering signals are observed due to side reactions of the matrix with alkali metal ions which can impair the analysis of the spectra.[2] 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. [3]

MALDI matrices used for identifying peptides include CHCA and DHB. These solutions are typically saturated solutions in a 50% Acetonitrile/0.1% TFA; TFA being the protonating agent. Typically the sample solution and matrix solution is mixed in a 1:1 ratio and then a half a microliter to a microliter is added to the MALDI plate. Proteins use SA (sinapinic acid) as their matrix; which is prepared in the same way as the peptide matricies.

Electrospray ionizationEdit

Electrospray ionization because of its principle of ionization is more demanding in the characteristics of the sample.[4] 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 nanoliter range; however, the detection limit of the instrument requires in protein analysis a relatively high concentration of the analyte.[citation needed] Therefore, usually a step to increase the concentration of the sample is needed.

The evaporation of the solvent in the spray during the ionization 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.[citation needed]

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.


  1. Fenyo D, Wang Q, DeGrasse JA, Padovan JC, Cadene M, Chait BT (2007). "MALDI sample preparation: the ultra thin layer method". J Vis Exp (3): 192. doi:10.3791/192. PMID 18978997. 
  2. Xu Y, Bruening ML, Watson JT (2003). "Non-specific, on-probe cleanup methods for MALDI-MS samples". Mass Spectrom Rev 22 (6): 429–40. doi:10.1002/mas.10064. PMID 14528495. 
  3. Smirnov et al., Anal. Chem., 76 (10), S. 2958-2965, 2004
  4. Du L, White RL (November 2008). "Improved partition equilibrium model for predicting analyte response in electrospray ionization mass spectrometry". J Mass Spectrom 44 (2): n/a. doi:10.1002/jms.1501. PMID 19003789.