Matrix assisted laser mass analyzers

Laser-desorption mass spectrometry (LDMS) or matrix-assisted laser desorption ionization (MALDI) coupled to a time-of-flight analyzer produces protonated or deprotonated molecular ion clusters for peptides and proteins up to masses of several thousand. 

Most biochemical analyses by MS use either electrospray ionization (ESI) or matrix-assisted laser desorption ionization (MALD1), typically linked to a time-of-flight (TOF) mass analyzer. Both ESI and MALDl are "soft" ionization methods that produce charged molecules with little fragmentation, even with biological samples of very high molecular weight. 

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)... 

Tandem mass spectrometry (MS/MS) is a method for obtaining sequence and structural information by measurement of the mass-to-charge ratios of ionized molecules before and after dissociation reactions within a mass spectrometer which consists essentially of two mass spectrometers in tandem. In the first step, precursor ions are selected for further fragmentation by energy impact and interaction with a collision gas. The generated product ions can be analyzed by a second scan step. MS/MS measurements of peptides can be performed using electrospray or matrix-assisted laser desorption/ionization in combination with triple quadruple, ion trap, quadrupole-TOF (time-of-flight), TOF-TOF or ion cyclotron resonance MS. Tandem... 

The focus of this chapter is the development of a technique often called wholecell matrix-assisted laser desorption ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) or whole-cell MALDI-TOF MS. Some groups prefer to use terms such as intact or unprocessed rather than whole, but the intended meaning is the same regardless of which word is used. As noted in the first chapter of this book, there are many different methods for the analysis of bacteria. However, for the analysis of intact or unprocessed bacteria, whole-cell MALDI-TOF MS is the most commonly used approach. This method is very rapid. MALDI-TOF MS analysis of whole cells takes only minutes because the samples can be analyzed directly after collection from a bacterial culture suspension. Direct MALDI MS analysis of fungi or viruses is similar in approach1,2 but is not covered in this chapter. MALDI-TOF MS of whole cells was developed with very rapid identification or differentiation of bacteria in mind. The name (whole cell) should not be taken to imply that the cells are literally intact or whole. Rather, it should be taken to mean that the cells that have not been treated or processed in any way specifically for the removal or isolation of any cellular components from any others. In whole-cell analysis the cells have been manipulated only as necessary to... 

The ability to resolve and characterize complicated protein mixtures by the combination of 2DLC and online mass spectrometry permits the combination of sample fractionation/simplification, top-down protein mass information, and bottom-up peptide level studies. In our lab, the simplified fractions generated by 2D(IEX-RP)LC are digested and analyzed using common peptide-level analysis approaches, including peptide mass fingerprinting (Henzel et al., 1993 Mann et al., 1993), matrix-assisted laser desorption/ionization (MALDI) QTOF MS/MS (Millea et al., 2006), and various capillary LC/MS/MS methodologies (e.g., Ducret et al., 1998). 

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]. 

Many diseases are characterized by the expression of specific proteins1 in some cases, malignant cells yield unique protein profiles when total cellular protein extracts are analyzed by proteomic methods such as two-dimensional gel electrophoresis or matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS).2 High-throughput proteomic studies may be useful to differentiate normal cells from cancer cells, to identify and define the use of biomarkers for specific cancers, and to characterize the clinical course of disease. Proteomics can also be used to isolate and characterize potential drug targets and to evaluate the efficacy of treatments. 

Figure 2.1 Mass spectrometric approach. Dl, direct inlet GC, gas chromatography HPLC, high performance liquid chromatography CZE, capillary zone electrophoresis El, electron ionization Cl, chemical ionization ESI, electrospray ionization DESI, desorption electrospray ionization APCI, atmospheric pressure chemical ionization MALDI, matrix assisted laser desorption ionization B, magnetic analyzer E, electrostatic analyzer...

Foret et al.98 collected fractions of model proteins and variants of human hemoglobins after fractionation by CIEF, and then analyzed them by matrix-assisted laser desorption-time-of-flight-mass spectrometry (MALDI-TOF-MS). As the authors point out, MS is an orthogonal method to CIEF because it separates according to molecular mass. 

The analysis for proteins present in plasma or a cell extract is a challenging task due to their complexity and the great difference between protein concentrations present in the sample. Simple mixtures of intact proteins can be analyzed by infusion with electrospray ionization and more complex ones by matrix assisted laser desorption ionization. MALDI is more suited for complex mixtures because for each protein an [M+H]+ signal is observed while for ESI multiply charged ions are observed. Surface enhanced laser desorption (SEEDI) is a technique for the screening of protein biomarkers based on the mass spectrometric analysis of intact proteins [49]. However in most cases for sensitivity reasons mass spec-... 

A toluene solution of polymer 15 was added to an aqueous mixture of peptides. After effective equUibration, the heterogeneous mixture was allowed to separate and the organic layer was analyzed by matrix assisted laser desorption ionization mass spectrometry analysis. Because the pH of the aqueous solution was 7.1, the peptides with pis above 7.1 were extracted and the peptides with pis below 7.1... 

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