MALDI mixtures

Peptide mass fingeiprinting (PMF) is a mass spectrometry based method for protein identification. The protein is cleaved by an enzyme with high specificity (trypsin, Lys-C, Asp-N, etc.) or chemical (CNBr). The peptide mixture generated is analyzed by matrix-assisted laser desorp-tion/ionization (MALDI) or electrospray ionization (ESI)... 

A major difference between MALDI and FAB is that a solid rather than a liquid matrix is used and a mixture of the analyte and matrix is dried on the laser target. For this reason, the effective combination of HPLC with MALDI is not as readily achieved although, since MALDI is largely free of the suppression effects experienced with FAB, it is able to provide useful analytical data directly from mixtures. 

The unseparated digest mixture was studied directly by mass spectrometry using matrix-assisted laser desorption ionization (MALDI) and this showed six of the polypeptides detected by LC-MS and three of the expected polypeptides that had not been detected by LC-MS. In contrast, MALDI did not show three polypeptides observed by LC-MS. 

MALDI-ToF is a technique that allows the molecular weights of proteins and peptides to be determined. It is less susceptible to suppression effects than electrospray ionization and thus is able to be used for the direct analysis of mixtures. In the case of a crude tryptic digest, MALDI-ToF will provide a molecular weight profile of the polypeptides present without the analysis time being extended by the need to use some form of chromatographic separation. 

Dai, Y., Whittal R.M., and Li, L., Two-layer sample preparation a method for MALDI-MS analysis of complex peptide and protein mixtures, Anal. Chem., 71, 1087, 1999. 

Figure 9.8 UV MALDI-MS spectrum of a mixture of polymer additives. After Jackson et al. [57]. Reprinted from A.T. Jackson et al., Rapid Communications in Mass Spectrometry, 10, 1449-1458 (1996). Copyright 1996 John Wiley Sons, Ltd. Reproduced with permission...

As noted above, whole-cell MALDI-TOF MS was intended for rapid taxonomic identification of bacteria. Neither the analysis of specific targeted bacterial proteins, nor the discovery of new proteins, was envisioned as a routine application for which whole cells would be used. An unknown or target protein might not have the abundance or proton affinity to facilitate its detection from such a complex mixture containing literally thousands of other proteins. Thus, for many applications, the analysis of proteins from chromatographically separated fractions remains a more productive approach. From a historical perspective, whole-cell MALDI is a logical extension of MALDI analysis of isolated cellular proteins. After all, purified proteins can be obtained from bacteria after different levels of purification. Differences in method often reflect how much purification is done prior to analysis. With whole-cell MALDI the answer is literally none. Some methods attempt to combine the benefits of the rapid whole cell approach with a minimal level of sample preparation, often based on the analysis of crude fractions rather... 

Figure 12.4 MALDI analysis of peptides prepared in situ from a 10 1 mixture of Bacillus cereus and Bacillus anthracis sp. Sterne.82 (a) Survey spectrum of peptide products. Species assignments are indicated on the figure. (b) Spectrum of fragment ions produced by low-energy collisions of the Bacillus cereus-specific peptide of mass 1529. (c) Spectrum of fragment ions produced by low-energy collisions of ions of the Bacillus anthracis peptide of mass 1528.

Both absolute quantitation and relative quantitation of species in mixtures is of interest in some circumstances. Quantitation in a 5-minute analysis can be achieved by addition of an internal standard, ideally the target microorganism grown in special media to incorporate heavy isotopes92-95 and determination of the relative peak heights of pairs of proteins from the analyte and the standard. Isotope-labeled proteins or peptides, selected to match proteins or peptides characteristic of target microorganisms, can also serve as internal standards for isotope ratio measurement. The addition of unmatched proteins or peptides is less reliable for either ESI or MALDI measurements because of unpredictable suppression in the variable mixture. 

The use of computer algorithms to deconvolute MALDI or ESI spectra of mixtures of microorganisms obtained under uncontrolled conditions is less likely to be successful, as discussed above. 

Warscheid, B. Fenselau, C. A targeted proteomics approach to the rapid identification of bacterial cell mixtures by MALDI mass spectrometry. Proteomics 2004, 4, 2877-2892. 

Warscheid, B. Jackson, K. Sutton, C. Fenselau, C. MALDI analysis of Bacilli in spore mixtures by applying a quadrupole ion trap time-of-fhght tandem mass spectrometer. Anal. Chem. 2003, 75, 5608-5617. 

MALDI-MS studies conducted on whole-cell bacteria prior to 2000 utilized spectra that rarely contained peaks above 20,000 Da. In 2000 a paper describing a methodology was published that extended the mass range for whole-cell bacteria to 100,000 Da.18 The matrix solvent consisted of a mixture of formic acid, acetonitrile, and water in a ratio of 17 33 50. The solvent mixture has been successfully used with most of the common MALDI matrices. The reasons for the success of this solvent are most likely related to the crystal formation of the matrix. When compared to other solvents, smaller and more uniform matrix-analyte crystals were observed with the method using formic acid, acetonitrile, and water as the solution. Figure 14.4 shows an example of E. coli obtained utilizing the described methodology. 

---