MALDI time-of-flight

Easterling, M. L. Colangelo, C. M. Scott, R. A. Amster, I. J. Monitoring protein expression in whole bacterial cells with MALDI time-of-flight mass spectrometry. Anal. Chem. 1998, 70, 2704-2709. 

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... 

Stump, M. J. Jones, J. J. Fleming, R. C. Lay, J. O., Jr. Wilkins, C. L. Use of double-depleted 13C and 15N culture media for analysis of whole cell bacteria by MALDI time-of-flight and Fourier transform mass spectrometry. J. Am. Soc. Mass Spectrom. 2003,14,1306-1314. 

Pittenauer, E. Quintela, J. C. Schmid, E. R. Allmaier, G. Paulus, G. de Pedro, M. A. Characterization of Braun s lipoprotein and determination of its attachment sites to peptidoglycan by 252-Cf-PD and MALDI time-of-flight mass spectrometry. /. Am. Soc. Mass Spectrom. 1995, 6, 892-905. 

Wenzel, R.J., Matter, U., Schultheis, L., Zenobi, R. (2005). Analysis of megadalton ions using cryodetection MALDI time-of-flight mass spectrometry. Anal. Chem. 77, 4329 1337. 

In principle, MALDI-TOF (MALDI-Time Of Flight) analysis allows for the determination of the complete polymer mass distributions and, from that, the calculation of various molecular weight averages like Mn and Mw and the... 

R. J. Wenzel, U. Matter, L. Schultheis, and R. Zenobi. Analysis of Megadalton Ions Using Cryodetection MALDI Time-of-Flight Mass Spectrometry. Anal. Chem., 77(2005) 4329-4337. 

M. W. F. Nielen. MALDI Time-of-Flight Mass Spectrometry of Synthetic Polymers. Mass Spectrometry Reviews, 18, no. 5 (1999) 309-344. 

For the quick characterisation of polydisperse surfactants with relative high molecular weight distributions matrix-assisted laser desorption/ionisation (MALDI)-time of flight (TOF)-MS represented an interesting alternative since low mass compounds did not interfere with the mass spectrometric detection of the compounds of interest. For example, the mass spectrum of C12-APG (Fig. 2.7.8) exhibited equally spaced signals with Am/z 162 corresponding to sodiated adduct ions of the mono- (m/z 371) to heptaglucosides (m/z 1343) [7]. 

For the characterisation of the biodegradation intermediates of C12-LAS, metabolised in pure culture by an a-proteobacterium, Cook and co-workers [23] used matrix-assisted laser desorption/ionisation (MALDI)-time of flight (TOF)-MS as a complementary tool to HPLC with diode array detection and 1H-nuclear magnetic resonance. The dominating signal in the spectrum at m/z 271 and 293 were assigned to the ions [M - H] and [M - 2H + Na]- of C6-SPC. Of minor intensity were the ions with m/z 285 and 299, interpreted to be the deprotonated molecular ions of C7- and C8-SPC, respectively. 

Partial mass spectrum of cow s milk (containing 2% milk fat) observed by MALDI/time-of-flight mass spectrometry. [From R. M. Whlttal and L Li, "Time-Lag Focusing MALDI-TOF Mass Spectrometry," Am. Lab. December 1997, p. 30.]... 

Another interface commonly used for connecting HPLC to a mass spectrometer is not a true in-line interface. It is a robotically controlled spotter plate system for collecting samples from the HPLC to be injected into the MALDI time-of-flight laser ionization mass spectrometer for analyzing proteins and large peptides. The effluent sample dropped in the plate well is mixed with an ionization matrix already present, solvent and volatile reagents are evaporated, and the plate is then placed into the injector target and blasted with a pulsed laser to volatilize and ionize sample into the atmosphere of the interface where it can be drawn into the mass spectrometer. 

Much of the pressure to develop automated sequential HPLC separations has come from the necessity to separate complex biological mixtures, especially protein mixtures. Traditionally, complex mixtures of proteins have been separated using two-dimensional gel electrophoresis (2D GEP). The first dimension gel separation is carried out with electrophoresis buffers, the gel plate is rotated 90° and the second SDS-PAGE separation is carried out under denaturing conditions, using sodium dilauryl sulfate. The separated spots are then visualized, scraped off the plate, and then extracted for further analysis. Protein analysis by MALDI time-of-flight mass spectrometry starts with this time- and labor-intensive 2D GEP separation mode. 

Matrix-assisted laser desorption/ionization (MALDI)-time-of-flight (TOF)-mass spectrometry (MS) is now routinely used in many laboratories for the rapid and sensitive identification of proteins by peptide mass fingerprinting (PMF). We describe a simple protocol that can be performed in a standard biochemistry laboratory, whereby proteins separated by one- or two-dimensional gel electrophoresis can be identified at femtomole levels. The procedure involves excision of the spot or band from the gel, washing and de-stain-ing, reduction and alkylation, in-gel trypsin digestion, MALDI-TOF MS of the tryptic peptides, and database searching of the PMF data. Up to 96 protein samples can easily be manually processed at one time by this method. 

Developments in mass spectrometry technology, together with the availability of extensive DNA and protein sequence databases and software tools for data mining, has made possible rapid and sensitive mass spectrometry-based procedures for protein identification. Two basic types of mass spectrometers are commonly used for this purpose Matrix-assisted laser desorption/ionization (MALDI)-time-of-flight (TOF) mass spectrometry (MS) and electrospray ionization (ESI)-MS. MALDI-TOF instruments are now quite common in biochemistry laboratories and are very simple to use, requiring no special training. ESI instruments, usually coupled to capillary/nanoLC systems, are more complex and require expert operators. We will therefore focus on the use of MALDI-... 

Fig. 1. Matrix-assisted laser desorption/ionization (MALDI)-time-of-flight (TOF) spectrum of a trypsin-digested one-dimensional gel band. Peaks are labeled with their monoisotopic masses. Note that these are not the masses of the peptides, but of the peptide (pseudo)molecular ions. In MALDI spectra, peptide molecular ions arise predominantly through the addition of a proton to the peptide, giving a mass increase of 1.007 Da. The molecular ions are usually denoted as MH+ or [M+H]+.

MALDI time-of-flight (TOF) MS was introduced in the late 1980s. This technique employs the co-crystallizing of matrix (low-molecular-weight organic crys-... 

Voyager DE MALDI time of flight (TOF) mass spectrometer (Applied Biosystems). 

MALDI-Fourier transform ion cyclotron resonance mass spectrometry MALDI-Q-ion mobility-TOFMS MALDI-Quadrupole-ion-trap-TOF mass spectrometry MALDI-TOF-imaging mass spectrometry MALDI Time-of-flight mass spectrometry Magnetic resonance imaging Matrix solution fixation Moxifloxacin... 

TMS) was used as a reference. Frequencies of proton measurements were 250 MHz, 300 MHz or 400 MHz and frequencies of carbon measurements were 50.3 MHz, 62.9 MHz, 75.5 MHz or 100.6 MHz. MS spectra were measured on a Fisons MALDI-time-of-flight (TOF) spectrometer or a Fisons Platform II ESI spectrometer. CNH elemental analyses were done on a Foss Heraeus CHN-O-Rapid device. 

Enzymatic assays can be applied in the marine environment to provide indirect information on dissolved compounds that are available to fuel bacterial production. Approaches that have been commonly appHed include measuring hydrolytic enzyme activities in seawater and monitoring degradation rates of model compounds. Protein hydrolysis in seawater is rapid as expressed by model protein studies (e.g., Nunn et al., 2003 Pantoja and Lee, 1999). This rapid and selective removal of dissolved proteins explains the relatively minor contribution from proteins to the accumulating DOM reservoir even though proteins are by far the most abundant intracellular biochemical. In an elegant study, Nunn and coworkers (2003) used matrix assisted laser desorption/ionization (MALDI) time of flight (TOP) mass... 

Although FAB has been used in polymer analysis, problems with fragmentation and the relatively low mass limit has made this less popular as new techniques have emerged. Plasma desorption has been used successfully but this too has waned in popularity with commercial spectrometers not really readily available. To a large extent polymer mass spectrometry equates to MALDI time-of-flight and the remainder of this article will bear this in mind. However, the use of electrospray ionisation (ESI) will be considered in conjunction with either quadrupole detectors or ion cyclotron resonance (ICR) N. B. ICR detectors can also be used with MALDI, as this is important and probably not as widely used as it could be. 

Fig. 1 Biomolecular interaction analysis/mass spectrometry (BIA/MS). Biosensor chips are derivatized with affinant (or used with an affinant of streptavidin) and used in the BIA analysis of biological fluids. The chips are then introduced into a MALDI time-of-flight mass spectrometer and retained ligands analyzed by virtue of molecular weight.

Next to electrospray ionization (Ch. 6.3), MALDI is the most important ionization technique in the MS analysis of biomacromolecnles. MALDI was introduced by Tanaka et al. [12] and Karas and Hillenkamp in 1988 [13], Today, MALDI plays an important role in the characterization of proteins, oligonncleotides, sugars, and synthetic polymers. MALDI-time-of-flight MS is an essential tool in the current proteomics research (Ch. 18.2). 

MALDI)-time-of-flight (TOF) MS (70), although the volume of data generated for a library by this technique can require gigabytes of disk storage space. 

C. MALDI Time-of-Flight Mass Spectrometer (MALDI-TOF-MS)... 

FIGURE 4.5 The five mass spectrometers commonly used for proteomic research, (a) ESI triple-stage quadrupole mass spectrometer (b) ESI quadrupole ion trap (QIT) mass spectrometer (c) MALDI time-of-flight mass spectrometer. 

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