IR-MALDI

Most often proteins are the bacterial biopolymers studied using MALDI MS either from fractions or whole cells. They are not the only isolated cellular biopolymers studied by MALDI, nor the first. Very soon after the introduction of MALDI there were a few reports of the analysis of bacterial RNA or DNA from bacterial fractions. One of the first applications of MALDI to bacteria fractions involved analysis of RNA isolated from E. coli,4 Other studies included analysis of PCR-amplified DNA,5 6 DNA related to repair mechanisms7 and posttranscriptional modification of bacterial RNA.8 While most MALDI studies involve the use of UV lasers, IR MALDI has been reported for the analysis of double stranded DNA from restriction enzyme digested DNA plasmids, also isolated from E. coli.9... 

D. Schleuder, F. Hillenkamp, and K. Strupat. IR-MALDI-Mass Analysis of Electroblotted Proteins Directly from the Membrane Comparison of Different Membranes, Application to On-membrane Digestion, and Protein Identification by Database Searching. Anal. Chem., 71(1999) 3238-3247. 

Note The vast majority of MALDI instruments use UV nitrogen lasers (337 nm, 3 ns). IR-MALDI has been restricted to applications where its deeper penetration offers advantages, e.g., for the direct desorption of analytes from sodium dodecyl sulfate (SDS) gels or thin layer chromatographic (TLC) plates. 

Leisner, A. Rohlfing, A. Berkenkamp, S. Rohling, U. Dreisewerd, K. HUlenkamp, F. IR-MALDI With the Matrix Glycerol Examination of the Plume Expansion Dynamics for Lasers of Different Pulse Duration. 36. DGMS Jahrestagung 2003, Poster. 

As alternatives to the cleavage of intermediates from a support and their characterization in solution, various methods have been developed for analyses of support-bound intermediates. The most common analytical tools include combustion analysis, colorimetric assays for specific functional groups, IR, MALDI-TOF MS, TOF-SIMS, and NMR. 

The ionization methods reported for IMS included MALDI [41,76-80], Secondary Ion Mass Spectrometry (SIMS) [19, 81-86], Matrix-enhanced (ME)-SIMS [87, 88], Desorption Electrospray Ionization (DESI) [89-99], Nanostructure Initiator Mass Spectrometry (NIMS) [100-102], Atmospheric Pressure Infrared MALDI Mass Spectrometry (AP-IR-MALDI-MS) [103], Laser Ablation-inductively Coupled Plasma-Mass Spectrometry (LA-ICP-MS) [104-106], Laser Desorption Postionization (LDPI) [107], Laser Ablation Electrospray Ionization Mass Spectrometry (LAESI) [108, 109], and Surface-assisted Laser Desorption/ioniza-tion Mass Spectrometry (SALDI) [110-112], Another method was called probe electrospray ionization (PESI) that was used for both liquid solution and the direct sampling on wet samples. 

MALDI spectra obtained with UV or IR lasers are essentially identical for most analysed samples. There are only very small differences. Indeed, when an IR laser is used, only less fragmentation is observed, indicating that the IR-MALDI is somewhat cooler. On the other hand, IR-MALDI induces a larger depth of vaporization per shot that leads to shorter lifetime of the sample. Compared with UV-MALDI, a somewhat lower sensitivity is observed. 

Common UV-MALDI matrices are listed in Table 1.2 with the class of compounds with which they are used. The matrices used with IR lasers, such as urea, caboxylic acids, alcohols and even water, are often closer to the natural solutions than the highly aromatic UV-MALDI matrices. In addition, there are many more potential matrices for IR-MALDI owing to the strong absorption of molecular compounds at IR wavelengths, even if the correlation between ion formation and matrix absorption in IR-MALDI is not clear [36],... 

Eckerskom, C., Stmpat, K., Kellermann, J., Lottspeich, F. and Hillenkamp, F. (1997) High-sensitivity peptide mapping by micro-LC with on-line membrane blotting and subsequent detection by scanning-IR-MALDI mass spectrometry. J. Protein Chem. 16, 349-362. 

Cramer, R. Burlingame, A.L. IR-MALDI—Softer Ionization in MALDI-MS for Studies of Labile Macromolecules, Mass Spectrometry in Biology and Medicines, pp. 289-307 in Burlingame, A.L. Carr, S.A. Baldwin, M.A., eds., Humana Press, Totowa, NJ (2000). 

Woods, A.S. et al., IR-MALDI-LDI combined with ion mobility orthogonal time-of-flightmass spectrometry, J. Preoteome Res., 5, 1484, 2006. 

Rohlfmg, A., Milthing, J., Pohlentz, G., Distler, U., Peter-Katahnic, J., Berkenkamp, S. and Dreisewerd, K., IR-MALDI-MS analysis of HPTLC-separated phospholipid mixtures directly from the TLC plate. Anal Chem, 79 (2007) 5793-5808. 

For the IR-MALDI off the blot, residues of blot buffer are disruptive. Thus, wash thoroughly Furthermore, the PVDF membrane must lie in the right orientation on the MALDI sample carrier. Because of the high binding capacity of the PVDF membranes, the protein binds only to the surface of the PVDF membranes, namely, on the blot side, where the gel came into contact with the membrane. Remember buttered side up. 

The IR-MALDI is not suitable for the analysis of peptide digestion by proteins. The digestion buffer probably does not go down well with the matrix. Thus, it would be ideal if you were able to measure with IR lasers as well as UV lasers in one device. 

In addition to UV/IR-MALDI, there is another possibility for bringing protein ions into the gas phase electrospray ionization (ESI). Figure 7.7 illustrates how the method works. The proteins are not gasified via incorporation into an evaporable matrix but by spraying the protein solution as finest droplets. Weak acids serve as ionization helpers, and organic solvents as spraying helpers. Acetonitrile/water 50 50 with 0.1% acetic acid is a typical carrier solution. Salts and detergents disrupt ESI and have to be removed. For this, Troxler et al. (1999) use small return-phase columns (Cg, elution with TFA, acetonitrile). 

IR-MALDI spectra show a lower mass resolution compared to that obtained by UV-MALDI. However, the IR-MALDl technique may be used for the analysis of halogenated polymers, which often show extensive fragmentation in UV-MALDl. ... 

Mass spectra of desalted Photofrin, in the positive ionization modes, obtained by (A) FAB/MS, (B) UV-MALDI/MS, (C) IR-MALDI/MS, (D) ESI/MS (nozzle-skimmer voltage 100 V). (E) Mass spectrum of per-methyl ester of Photofrin, in the positive ionization mode, obtained by LD/... 

MALDI is the dominant ionization source choice for MSI however, it is not without drawbacks. The application of MALDI matrix on top of the tissue surface complicates the analysis by adding potentially isobaric matrix cluster ions that may obscure the drug compound. Careful selection of MALDI matrix can reduce the matrix effect. An alternative would be to either use the water native to the tissue as a matrix such as with IR-MALDI or altogether eliminate the need for matrix. DESI is an atmospheric pressure technique that permits the direct analysis of surface samples, including tissue sections, with minimal sample preparation (Takats et al., 2004 Cooks et al., 2006). In contrast to MALDI—MSI, no matrix is required however, the spatial resolution for DESI—MSI is worse when compared to MALDI or SIMS imaging experiments. 

Eor IR-MALDI, the situation can be very different because of the larger penetration depth, resulting in a larger acoustic time constant of about 1 ns. Eor the desorption with an Er YAG laser, the pulse width of 100 ns is long compared to... 

Figure 1.2 High-speed time-lapse photographs of IR-MALDI plumes generated with an optical parametric oscillator laser with 6-ns pulse width (left panels) and an Er YAC-laser with 100-ns pulse width (right panels). Both lasers were operated at 2.94 4m wavelength. Matrix, glycerol time resolution, 8 ns spatial resolution, 4 im. The top three panels represent gradients of gaseous material density creating gradients...

IR-MALDI MALDI with infrared laser wavelengths... 

Figure 5.4 IR-MALDI mass spectrum of large DNA analyzed from glycerol as a matrix. The spectrum depicts single-stranded DNA molecules of length up to 1.4 kb generated from restriction-digested plasmid DNA purified by ethanol precipitation. The...

Figure 5.8 IR-MALDI reflectron TOF-mass spectrum of a mixture of double-stranded DNA fragments recorded from a glycerol/ ammonium acetate matrix. The sample was generated by digestion of the plasmid...

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