Mass Spectrometry

The Vestec Electrospray Spectrometer is a unique investigative tool for our laboratory. Its ionization technique is soft and non-destructive thus allowing one to acquire spectra of proteins, peptides, and other bipolymers with virtually no fragmentation of the parent molecule. Its effective mass range is up to 60 kDaltons due to the fact that most biopolymers become multiply charged when exposed to an electrical potential of a few kilovolts. Spectral data interpretation is relatively straightforward and quick, with sample preparation requires a minimum number of steps (<10 minutes from solvation to spectral acquisition). The buffer system used in the instrument is simple and compatible with HPLC and a variety of other separation techniques, thus offering the possibility of easy interfacing to other laboratory instrumentation. Another advantage of the electrospray ionization technique is that is non-selective with respect to the primary structure of the protein/peptide analyzed. Thus one can measure the molecular weight of a large number of proteins regardless of whether they are post translationally modified or not (i.e. disulfide bridged, glycosylated, etc.) with an accuracy as high as 0.01%. this is in contrast with traditional SDS/PAGE separation technique which, besides the fact that it only has a 5-10% accuracy in determining molecular weights of proteins, is subject to complications when separating proteins whose mass does not correlate well with their migration distance in the gel (i.e. glycoproteins are known to migrate differently than the mass calibration markers). The fact that the protein/peptide ions are in gas phase when detected is an additional advantage of the technique because it eliminates the complexity associated with the solution phase behavior of these macromolecules such as conformational changes, aggregation, and other complicating factors.
We routinely employ the ESMS instrument in the measurement as a quality control tool for the verification of the structural integrity of our synthetic peptides. Unlike other current techniques such as Edman degradation, HPLC, and amino acid composition analysis which are rather slow and detect fewer products of synthesis, electrospray ionization offers an absolute confirmation that the peptide synthesized is the intended product. Problems such as deletions, chemical modifications, and incomplete removal of protective groups during the synthesis or cleavage/deprotection protocols are readily detected on amounts of sample as small as 1 picomole in total analysis times of less than 15 minutes.

Matrix Assisted Laser Desorption Ionization Mass Spectrometry

During the past five years, electrospray and matrix-assisted laser desorption ionization (MALDI) have emerged as powerful ionization techniques enabling a wide range of biologically important material to be identified by mass spectrometry. We have found that the strengths and weaknesses of both techniques complement each other well in obtaining mass data on biological compounds. One of the particular strengths of MALDI is its extraordinary tolerance to contaminants such as buffer salts and surfactants. This ability to tolerate contaminants sets MALDI apart, in marked contrast, from other desorption techniques.
The most numerous applications of both techniques have been applied to the
study of proteins. Dramatic examples of the application of MALDI to the analysis of crude biological fluids such as blood plasma and milk can be found in the literature. Researchers have succeeded in obtaining spectral data from protein samples present at a concentration of only 10-5 M in 10% solution sodium dodecyl sulphate (SDS) using a matrix of 2,5-dihydroxybenzoic acid. This tolerance to contaminants makes MALDI an excellent choice for preliminary screening experiments on biological extracts, enzymatic digest mixtures, fermentation broths, and similar complex mixtures.

General Guidelines For Mass Spec Sample Requirements

1) The protein/peptide should be dissolved in pure water or shipped completely dry if possible. Avoid sending samples in high ionic strength buffers as relatively low salt concentrations might interfere with the ionization process.
2) Any amount of sample can be submitted for analysis. Due to the fact the ionization efficiency and the detector sensitivity of the instrument are sample dependent it is rather hard to specify a minimum amount of sample that can be analyzed.
3) If the sample is shipped in a solution containing salt try to estimate the concentration of the salt. Also, providing a list of cautions and anions that might be present in the solution is extremely helpful because sometimes during the ionization process ions other than the customary proton become adducts as documented by recent studies (i.e. sulfate, phosphate, K+, Na+, etc). Of course, other types of information such as expected/theoretical amino acid sequence are very helpful.