Linear mode-MALDI

MALDl-TOF mass spectrometry (MS) has also been used to characterize PAM AM dendrimer composition with and without added Cu + [98]. linear-mode MALDI-TOF mass spectra of G2 and G3, and their complexes with Cu + ions, are shown in Fig. 9. 

MALDI-TOF mass measurement of the ball-type Pcs is the most powerful technique in order to determine their molecular weight and to characterize their structures. In this technique, it is important to find the appropriate matrix in order to obtain highly resolved spectra, thereby obtaining good mass resolution. If we take into account the MALDI-TOF-MS of the ball-type Pcs 33, 34, 35, and 36 in Fig. 9 [46] and 39 and 40 in Fig. 10 [47] as examples, the protonated molecular ion peaks of 33 and 34 were observed at low intensity, but the sodium adduct peaks of Pcs 33 and 34 were found to be dominant, intense peaks in the linear-mode MALDI-TOF-MS by using... 

Fig. 22 The positive ion and linear mode MALDI-MS spectrum of 39 in ACCA (15 mg/mL 1 1 water-acetonitrile) MALDI matrix using nitrogen laser accumulating 50 shots. Inset spectrum shows expanded mass region of 39 [47]...

Figure 6.3 Linear mode (-) MALDI spectra of a sialylated tetra-antennary N-glycan mixture obtained with (a) ATT/DAC (15gL 20mM) and (b) THAP/DACA (20gL 20mM) before and (c) after purification with graphitized carbon. Reprinted with permission from Ref [42],...

Figure 6.8 Linear mode MALDI spectra of native N-glycans obtained by in-gel PNCase F digestion from the 2-D gel of al-antitrypsin spots of a CDC-type-llx patient in the...

Figure 6.2 Whole-cell MALDI-TOF spectra of two Vibrio species taken using a modern TOF mass spectrometer. Even in the linear mode V. parahaemolyticus can be easily differentiated from V. vulnificus.

TOF analyzers are especially compatible with MALDI ion sources and hence are frequently coupled in aMALDI-TOF configuration. Nevertheless, many commercial mass spectrometers combine ESI with TOF with great success. For proteomics applications, the quadrupole TOF (QqTOF) hybrid instruments with their superior mass accuracy, mass range, and mass resolution are of much greater utility than simple TOF instruments.21,22 Moreover, TOF instruments feature high sensitivity because they can generate full scan data without the necessity for scanning that causes ion loss and decreased sensitivity. Linear mode TOF instruments cannot perform tandem mass spectrometry. This problem is addressed by hybrid instruments that incorporate analyzers with mass selective capability (e.g., QqTOF) in front of a TOF instrument. 

The mass accuracy is highly dependent on the mode the instrument is operating in. In the reflector mode, with time-lag focusing, the best MALDI-TOF and oa-TOF instruments are capable of achieving <5 ppm with internal standards, provided that the isotopes are resolved. In many cases it is not possible to add internal calibrants, and then the error in mass accuracy is often increased to 50-100 ppm. Operation of an instalment in a linear mode will typically decrease the mass accuracy. 

Fig. 10.6. Linear mode positive-ion MALDI-TOF spectra of ribonuclease B in 80 mM urea, (a) 300 fmol in CHCA, (b) 600 fmol in DHB, and (c) 300 fmol in CHCA/DHB matrix mix. Reproduced from Ref. [71] by permission. Elsevier Science, 2003.

In general the commercial TOP instruments have two detectors one for the linear mode and one for the reflectron mode. The combination of MALDI with TOP is ideal because both techniques are pulsed techniques. However, it is also possible to arrange a continuous beam as generated by electrospray ionization. Por that purpose orthogonal acceleration was developed [65]. The ion beam is introduced perpendicularly to the TOP and packets are accelerated orthogonally (oa-TOP) at similar frequencies improving the sensitivity. While a packet of ions is analyzed, a new beam is formed in the orthogonal acceleration. 

Select Monoisotopic if the MALDI data were acquired in reflectron mode, or Average for data acquired in linear mode. 

For the ZnPc 38, a linear-mode positive-ion mass spectrum could be obtained only in ACCA matrix even though other MALDI matrices were tested. The protonated molecular ion peak of 39 was observed at 2,315 DA, which coincided with the theoretical value. Besides the protonated molecular ion peak, no other peak was... 

MALDI mass spectra were acquired on a Broker Reflex mass spectrometer equipped with a 337 nm nitrogen laser and a multiple sample stage source. The spectra were acquired in linear mode, represent the sum of 20 laser shots, and are un-smoothed. All ions were desorbed at a laser power just above threshold, at an ion extraction voltage of 30 kV. The matrix used was a saturated solution of 3,5-dimethoxy-4-hydroxycinnamic acid (sinapinic acid, Aldrich Chemicals) in a 1 1 water/acetonitrile solution. Low-mass matrix ions were deflected by the application of a voltage pulse. 

The increased mass accuracy available when the instrument was operated in the reflectron mode was important for the analysis carried out. For example, fractions 4b and 7 or fractions 5 and 8 from C. ermineus appeared to be the same species when measured with the instrument operated in the linear mode. Only in the reflectron mode were we able to reliably distinguish the masses of each species. The high sensitivity of MALDI-TOF is particularly important for the analysis of native peptides such as conotoxins where often the venom of many milkings must be collected to obtain sufficient material for sequence analysis. The increased sensitivity of MALDI over LSIMS is illustrated in the analysis of fraction 5 from C. striatus venom (see Table I). Despite the two orders of magnitude difference in the amount of material consumed in the LSI experiment we did not discern any intact species in fraction 5, whereas the MALDI measurement yielded useful information. However, the comparisons in Tables I and II reveal that some components may be detected by LSIMS but not observed in the MALDI mass spectrum (measured with any of the matrices or sample preparation methods). The contrary is most likely more prevalent, i.e. that a large number of the species detected by MALDI with one or more of the matrices are difficult species to ionize with LSIMS. 

MALDI-TOF was done on a Kratos Kompact Maldi III mass spectrometer fitted with a standard 337 nm nitrogen laser and operated in the linear mode at an accelerating voltage of 20 kV. The matrix used was a-cyano-4-hydroxyciimamic acid (33mM in acetonitrile/methanol, premade from BRS) at a ratio of 1 1 with purified peptide samples. MALDI sample slides were loaded with O.S-1.0 uL of matrix/sample mixture (estimated 1-10 pmol peptide). The data was reprocessed using the Kratos software provided with the instrument. Theoretical masses were determined by utilizing a spreadsheet in which individual peptide masses were added to all possible caibohycfrate forms these masses were then compared to the observed masses to identify structures consistent with the mass results obtained. 

MALDI-MS was performed using a Kratos Kompact MALDI III mass spectrometer fitted with a standard 337 nm nitrogen laser, and operated in the linear mode at an accelerating voltage of 20 kV. Two sample preparation methods were used (1) for collected peptides, 0.3 pL aliquots of sample and matrix (a-cyano-4-hydroxy cinnamic acid, Biomolecular Separations, Inc.) were mixed on the probe slide and allowed to air dry or (2) for unfiactionated digests, a thin polycrystalline film was prepared according to (11) (with modifications for use on a probe slide (12)), matrix and sample aliquots were mixed (usually 0.3 pL each) on this surface and prior to drying, rinsed twice with 2 pL of deionized water. 

ESMS was perfonned with a Fisons VG Quattro outfitted with a Hsons Electrospray Source. Samples were dissolved in 1.0 mL of 50% methanol-1% acetic acid, then diluted 1 10 with 50% acetonitrile-1.0 mM anmumium acetate to give 25 pmol/pL. A 10 pL aliquot of each sample was injected into a 10 pL/min stream of 50% acetonitrile-1.0 mM ammonium acetate. Data was processed using Fisons MassLynx Software. MALDI-MS was performed with a Vestec Benchtop lit linear dme-of-flight mass spectrometer, opmted in the linear mode with an N2 laser (337 nm). Samples were dissolved in 1.0 mL of 25% acetonitrile-0.1% TFA, then diluted 3 100 to give 5-10 pmol/pL. A 0.5 pL aliquot of each sample solution was added to 0.5 pL of matrix [a-cyano-4-hydroxycinnamic aci saturated solution in 50% acetonitrile-2% TFA]. Samples were dried at ambient temperature and pressure. Each spectrum was the sum of ion intensity from 10-50 larer pulses. Tlie mass axis was calibrated externally. 

All mass spectra were acquired on a Kratos Kompact MALDI III time of flight mass spectrometer with a 337 nm N2 laser and a 20 kV extraction potential in the linear mode. Membranes were affixed to the Kratos sample slide with tape. Every spectrum was the average of 50 laser shots. Spectra were calibrated from external standards desorbed from the same membrane being tested. 

Matrix Assisted Laser Desorption Ionization-Mass Spectrometry (MALDI-MS) Mass spectra of native and denatured antibodies were obtained with a PerSeptive Biosystems (Farmingham, MA.) Voyager Elite mass spectrometer operated in the linear mode with a Laser Sciences Inc., 337 nm nitrogen laser. hAB-1 was denatured by boiling the sample in 1.0 M guanidine-HCl, 50 mM Tris pH 7.5 buffer. Native and denatured samples were diluted with 20 mM Tris, 10 mM octylglucoside (Tris/OG) pH 6.8 buffer prior to MALDl-MS analysis. Proteins were spotted on the sample plate as a sandwich between two layers of the matrix. The bottom layer consisted of 100 mM sinapinic acid in acetonitrile and the top layer consisted of 50 mM sinapinic acid in 30% acetonitrile / 70% H2O / 0.07% TFA. The m/z scale of the instrument was calibrated using a Hewlett-Packard protein standard mixture. 

Figure 7.11 MALDI spectra of seed proteins extracted from Raboso Piave (a), Prosecco (b), and Malvasia Nera di Brindisi (c) (Measurements in the positive ion linear mode of ions formed by a pulsed nitrogen laser at X = 337 nm with repetition rate 50 psec, ion source voltage 1 25 kV, ion source voltage 2 23,35 kV, ion source lens voltage 10.5kV. 5 p,L of sample mixed with 5 p,L of DBH matrix solutions, 1 p,L of mixture deposited on the stainless-steel sample holder). (Pesavento et al., JMS 2008 in press, DOI 10.1002/jms.l295)...

FIGURE 4.4 MALDI-TOF mass spectrum acquired using a Voyager DE-STR (PerSeptive Biosystems) in linear mode of four peptides the mixture consisted of 1 pmole/ rL each of des-Arg -bradykinin, = 903.47 angiotensin I, = 1295.68 Glu-fibrin-... 

For monomeric peptides, the mass is confirmed by MALDI MS using ce-cyano-4-hydroxydnnamic acid as the matrix using a Voyager DE Pro instrument in reflector mode. MALDI Mass Spectra of tetrameric peptide is obtained in linear mode using sinapinic acid as matrix. 

The MALDI-TOF-MS analysis of the polyMMA, obtained in linear mode shows only one series of peaks, whose interval was regular, ca. 100, the molar mass of MMA unit. It indicates the absence of irreversible chain termination processes via recombination or disproportionation. According the proposed mechanism of polymerization the absolute masses of the peaks should be equal... 

Fig. 2. Mass spectra (m/z 1000-10,000) acquired by MALDI-TOF/TOF MS in linear mode using serum sample fractionated by IMAC-Cu and hydrophobic C8 magnetic beads. The serum sample was fractionated using IMAC-Cu magnetic beads first and then the unbound solution was transferred to C8 magnetic beads for additional fractionation (tandem chromatography). Different profiles are generated using this tandem chromatography method.

We found that target preparation choices such as type and concentration of matrix, the ratio of analyte to matrix, on-target washing, and recrystallization procedures can dramatically affect the quality of resulting MALDI spectra. We usually try two different analyte to matrix dilutions 1-9 pL and -A pL (analyte to matrix) to ensure good-quality spectra. We tune the linear mode method in... 

Mass spectrometer There are several commercially available MALDI-MS apparatus. These have been recently reviewed m the section product review of Analytical Chemistry (497 A, 1995). The analysis of the relatively complex peptide profiles of nervous tissue requires a high resolution mass spectrometer, i.e., an apparatus equipped with a reflectron (or m the linear mode with a capacity for delayed ion extraction). 

In MALDI-TOP, the packets of ions produced by laser irradiation of matrix/ analyte mixture and accelerated by a fixed electric potential (V) can be detected in two different ways the linear and reflection (or reflectron) mode. The two detection modes are complementary, since a higher resolution is obtained in the reflection mode and a higher sensitivity at high molar mass is achieved in the linear mode. 

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