MALDI-ToF MS measurements

The first step in biopharmaceutical development is the selection of a clone in a specific cell line. Whole-mass analysis, if possible, is a fairly simple and powerful tool at this stage to verify the successful expression and translation of the desired protein. VanAdrichem et al.65 described the use of MALDI MS to monitor protein expression in several mammalian cell lines like CHO DXB11, CHO SSF3, and hybridomas. Quantitative MALDI-TOF MS measurements of an IgG antibody and insulin during large-scale production in hybridoma cells were comparable to affinity chromatography results. 

Early attempts to develop patterns of specific compounds and proteins were based on tissue homogenates (Bums, 1982 Jimenez et al., 1997 Mitchell et al., 1997). Tissue profiling, or more accurately called tissue imaging, based on direct mass spectrometry measurements is a much younger area of research which started being developed in late 90s when MALDI-TOF-MS measurements were performed on intact tissue sections (Chaurand et al., 1999). 

The MALDI-TOF MS data indicated that the optimum pH was 7.5. The amount of proteins, such as peak a of Fig. 7, can be estimated fairly accurately by taking the MALDI-TOF MS several times (136). Based on 10 different MALDI-TOF MS measurements, it appeared that about one-half of peptide deformylase was cleaved on incubation with 0.19 equiv of the catalyst for 72 h (Fig. 7), which corresponds to ko of 0.010 h Here, the initial fraction of peptide deformylase complexed with the catalyst cannot exceed 20%. The ko observed when peptide deformylase is fully bound to the catalyst is cat- The lower limit of at is, therefore, estimated as 0.050 h For the myoglobincleaving catalyst Co BU, fecat was 0.022 under the same conditions (126, 127). 

The activity of the catalyst candidates for angiotensin cleavage was initially screened by measuring MALDI-TOF MS after incubation of angiotensin I (5o = 0.02 mM) with the catalyst candidates (Co = 0.2 mM for each catalyst candidate assuming 100% overall synthetic yield) at 37°C and pH 7.5 for 24 h. Whether smaller fragments were formed was checked by the MALDI-TOF MS measurement. 

H-NMR analyses were performed using a Varian 400 MHz spectrometer at room temperature. Samples were dissolved in CDCI3 (5 wt%). MALDI-TOF MS measurements were carried out on a Voyager-DE-STR instrument (Applied Biosystems, Foster City, CA, USA). Tra 5-2-[3-(4-teit-butylphenyl)-2-methyl-2-propenylidene] malononitrile was used as the matrix, which was dissolved in tetrahydrofuran (THF) at a concentration of approximately 40 mg/ml. Sodium trifluoroacetate (Aldrich, 98%, Milwaukee, WI, USA) was used as the cationization agent and was added to THF at a concentration of 5 mg/ml. Samples were dissolved in THF at a concentration of 2 mg/ml. In a typical measurement, the matrix, cationization agent and sample solution were premixed in a 5 1 1 ratio. Approximately 0.5 pi of the mixture obtained was hand-spotted on the target plate and left to dry. Mass spectra were recorded in the reflector mode. 

This research was sponsored by DSM Research, The Netherlands. We thank Dr. E. Currie and Dr. J. Thies (DSM Research) for beneficial discussions and Marion A. van Straten for MALDI-ToF MS measurements. 

In 2014, Papagiamiitsis et al. described a new assay for the identification of CMY-2-like P-lactamases in clinical enterobacterial isolates by MALDI-TOF MS. The new method is based on the extraction of periplasmic proteins and the detection of CMY-2-like p-lactamases by MALDI-TOF MS according to their molecular weight. Successful extraction of p-lactamases from the periplasmic space of bacteria was a crucial step for the performance of the described assay. Therefore, a modified sucrose method for the extraction of periplasmic proteins has been used. Purified P-lactamases were used as positive controls for MALDI-TOF MS measurements, and for calibration and setting up the mass spectrometer s parameters. 

A modified version of 2DE and gel image analysis, with silver staining, autoradiography, and protein identification and measurement of peptide mass, uses matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) as a rapid and sensitive technique for identifying peptides. MALDI-TOF-MS applies well to protein detection in biological fluids.56 A second advantage of this technique is... 

Biomolecular MS and in particular MALDI-TOF-MS (see Sections 2.1.22 and 2.2.1) permit the routine analysis of oligonucleotides up to 70-mers, intact nucleic acids, and the direct detection of DNA products with no primer labels with an increase in analysis speed and mass accuracy especially in contrast to traditional DNA separation techniques such as slab gels or capillary electrophoresis. Applications focus on the characterization of single nucleotide polymorphisms (SNPs) and short tandem repeats (STRs). Precise and accurate gene expression measurements show relative and absolute numbers of target molecules determined independently of the number of PCR cycles. DNA methylation can be studied quantitatively. 

Bartolini, W.P. Johnston, M.V. Characterizing DNA Photo-Oxidation Reactions by High-Resolution Mass Measurements With MALDI-TOF-MS. J. Mass Spectrom. 2000,35,408-416. 

The product was identihed by a number of spectroscopic methods. Dioxygen uptake was measured by spectrophotometric titration. MALDI-TOF-MS (matrix-assisted laser desorption/ionization-time of flight-mass spectrometry), an MS method particularly suited to determining molecular masses of biopolymers and synthetic materials with relative masses up to several hundred kilodaltons, determined that the product contained stoichiometric amounts of the heme starting material, the copper complex, and dioxygen in a 1 1 1 ratio. 

In a time-of-flight (TOF) analyzer the time of flight of ions between the ion source and the detector is measured [61]. This requires that the time at which the ions leave the ion source is well-defined. Therefore, ions are either formed by a pulsed ionization method or various kinds of rapid electric field switching. The single discontinuous laser pulses at distinct time points used in MALDI can be ideally combined with time-of-flight mass separation. TOF analyzers thus received increasing interest with the development of MALDI MS. The schematic draw of a linear MALDI-TOF MS is shown in Fig. (9). 

Exact mass measurement at high resolution is an important tool along with other spectroscopic methods to help confirm the structure of novel flavonoids. It is used as structural proof when elemental analysis is not possible, e.g., when studying minor components. When EI-MS can be used, 1 to 10 pmol samples are required for one measurement however, when FAB-MS is used, 0.1 to 1 nmol is normally required. The use of ESI on a double focusing mass spectrometers and MALDI-TOF-MS requires smaller amounts of sample, and subpi-comole amounts of flavonoids may be adequate. 

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