LC and MALDI MS

In this chapter, the interfacing of LC and MALDI-MS and -MS/MS will be described, performance will be discnssed and selected applications in the proteomics field including analyses of membrane proteins will be presented. 

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Mukhopadhyay, R. (2005) The automated union of LC and MALDI MS. Anal. Chem. 77 150A-152A. 

Wehr, T. (2003). Coupling liquid-phase separations and MALDI-MS. LC-GC North Am. 21, 974—982. 

J. B. Young and L. Liang, An impulse-driven liquid-droplet deposition interface for combining LC with MALDI MS and MS/MS, J. Am Soc. Mass Spectrom., 17 (2006) 325-334. 

Figure 3. LC-MALDI strategy. Fractionation of the peptide mixture and MALDI-MS analysis.

The currently most frequently applied method for LC-MALDI-MS is automated post-column fractionation and on-plate collection in discrete spots of the LC colunm effluent. After the solvent is evaporated, the matrix solution can be added, and MALDI-MS analysis of the various spots can be performed. The procedure requires a liquid-handling robot, capable of disposition of effluent fractions at discrete spots on the MALDI target. A number of ways were proposed for deposition in discrete spots on the MALDI target, e.g., blotting via direct contact between droplet and target [139-140], piezoelectric flow-through microdispensing [141], pulsed electrical-mediated droplet deposition [142], and a heated droplet interface [143], Commercial LC-MALDI-MS devices were recently reviewed [144],... 

T.J. Tegeler, Y. Mechref, K. Boraas, J.P. Reilly, M.V. Novotny, Microdeposition device interfacing capillary electrochromatography and microcolumn LC with MALDI-MS, Anal. Chem., 76 (2004) 6698. 

Lastly, MS is compatible with high-throughput analysis. Each analysis, for both ESI-MS and MALDI-MS, is fast and lasts for less than a minute. As already mentioned, MALDI-MS analysis can easily be automated with dedicated software so that hundreds of samples can be analyzed without human intervention. ESI-MS lends itself less to high-throughput analysis as sample preparation and introduction in the ionization capillary are more tedious and time consuming. Still, automation is fully conceivable when coupled to LC for instance. 

Fig. 7. Application in practice. The spots of both gels are cut out after 2D-PAGE and Coomassie G-250 staining. The protein spots of one gel are analyzed by on-line nano-LC-ESI-MS/MS after tryptic digestion (a), whereas protein spots of the other gel are analyzed using off-line nano-LC and MALDI-TOF-MS/MS analysis (b).

MALDI-MS has mainly been used as an off-line detector for various LC separation techniques (iso-cratic and gradient HPLC, LCCC, SEC) of polymers. The problems of coupling of LC and MALDI-ToFMS are related to the fact that MALDI-ToF is based on the pulsed desorption of molecules from a solid surface layer and, therefore, a priori not compatible with liquid chromatography. Different semion-line interfaces have been developed for automated LC sample collection and subsequent preparation of the samples for MALDI-ToF analysis. In... 

The suspension of microbial cells in a solvent such as aqueous acidic acetonitrile is a procedure used routinely in soft ionization mass spectrometric investigations of microorganisms. This is particularly the case in MALDI-MS studies where whole-cell suspensions have been analyzed directly without separating the cellular residue. By contrast, ESMS is usually carried out with cell-free supernatants after analyte separation by LC. Some workers71 report that partial lysis of the cells occurs due to the acidic conditions employed in such techniques and that this results in the release of proteins and peptides from... 

LC-CC/MALDI-ToF-MS showed that the additional peak observed in the LC-CC chromatogram was PC with one f-butyl end-group and one OH end group no evidence for other end-groups was found ... 

The most discriminating technique for proving the identity and purity of analyte peak of a chromatogram, especially for analyzing biological samples and natural products, is by using online LC-UV/MS or GC-MS/FTIR methods [15]. Alternatively, one could use a combination of TLC and MS, where direct determination on the TLC plates is made by matrix-assisted laser desorption ionization mass spectrometry (MALDI MS) [16]. 

If no references for the degradation products or impurities are available in the laboratory, the sample should be exposed to stress conditions such as heat (50-80 °C), ultraviolet light (2000 lux), acid and base (0.1-1 M HC1 and NaOH), and oxidant (3% H2O2). After incubation in the allotted time, the purity and identity of the analyte peak/spot should be proved by using DAD or MS detection (for LC), MS (for GC), or in situ UV-Vis measurement using a densitometry or TLC-MALDI MS (for TLC). 

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