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Analysis of Phosphopeptide Using TOF-TOF

Phosphorylation is a type of post-translational modification of proteins that is used for the intracellular signal transduction in a wide range of living species. For this reason, it is very important to determine where the protein (amino acid) is phosphorylated. In this work, we measure a monophosphopeptide (FQ pS EEQQQTEDELQDK) that was obtained from the tryptic digestion of β-casein (Bovine) using the TOF-TOF option that is available for the JEOL Spiral- TOF™ system.

Analysis of organic compounds on an acrylic plate using JMS-S3000 "SpiralTOFTM"

Industrial materials are often evaluated by surface analysis instruments that provide information on surface elements, bonding states, and functional groups. However, there are limited options for surface analysis techniques that provide molecular weight and molecular structure information for organic compounds present on surfaces. Matrix Assisted Laser Desorption Ionization - Time of Flight Mass Spectrometry (MALDI-TOFMS) is a soft ionization technique that can be used to analyze surfaces in order to estimate elemental compositions with accurate mass measurements, obtain structural information by using MS/MS, and map surface compounds by using MS imaging. MALDI-TOFMS uses a high voltage on the target plate to accelerate the ions into the TOFMS analyzer. Therefore, the target plates are conductive and are typically made of stainless steel. MALDI imaging mass spectrometry is widely used for analyzing organic substances on frozen tissue sections. In this case, a frozen tissue section with a thickness of about 10 μm is placed on a conductive glass slide coated with an indium tin oxide (ITO) film. However, for the analysis of industrial products, the target organic compounds are on nonconductive substrates such as resins with millimeter thicknesses. MALDI-TOFMS surface measurements using nonconductive substrates lead to a reduction in mass resolution and a significant decrease in ion intensity due to surface charging. This problem can be solved by pretreating the surface with gold vapor deposition in order to change it from nonconductive to conductive. This method was previously shown to work well in MSTips No. 204 in which the gold vapor deposition method was applied to the MALDI-MS imaging analysis of inks on paper. In this report, we used gold vapor deposition to look at samples on the surface of a 1 mm thick acrylic plate.

“Remainders of KM” plot for polymers using msRepeatFinder: compositional mapping over a broad mass range

Mass Spectrometry (MS) with soft ionization such as matrix-assisted laser desorption ionization (MALDI) allows the compositional analysis of polymers (repeating units, chain terminations) of low dispersity. Combining a size exclusion chromatography (SEC) fractionation with a high-resolution MALDI SpiralTOF™ MS analysis enables the evaluation of the composition of polydisperse polymeric samples over a broad mass range (high-resolution/high-accuracy mass measurements in the low mass range < 4 kDa, isotopic resolution in higher mass range < 30kDa). However, as the resolution gets higher, more peaks are detected in the mass spectrum for each fraction, making the interpretation of the mass spectral data the rate-limiting step for the whole analytical procedure. In this work, a “remainders of Kendrick mass” (RKM) analysis is used as a rapid post-acquisition data processing tool that uses visual maps from combined low/high-accuracy and low/high mass range data.

The Relationship between Crystal Condition and Mass Resolving Power, Mass Accuracy

The JMS-S3000 SpiralTOF™ has a unique 17 m flight path that offers the highest resolution MALDI-TOF MS system currently available. With an extended flight distance, the SpiralTOF reduces topographic effect of matrix crystal to a minimum and achieves highly reproducible mass resolving power and high mass accuracy with external mass calibration. In this work, we demonstrate the measurement of a polymer standard with 4 types of matrices that are typically used for MALDI polymer measurement by using the JEOL SpiralTOF system. Additionally, we looked at the crystal condition using the JEOL JSM-7600F thermal field emission scanning electron microscope (FE-SEM).

Measurement of synthetic polymers: Polystyrene

Polystyrene (PS)1000 and 2400 were measured using the JMS-S3000 SpiralTOF. The [M+H]+ peaks of PS with the basic monomer units of 104u (Fig.1) were observed for each sample. The mass spectrum of PS1000 and an expanded view around m/z 1000 are shown in Fig. 2. The resolving power at m/z 1101 was approximately 50,000 (FWHM). The mass difference between 8, 9 and 10-mers showed a very good match with the theoretical mass number (104.0626) calculated from the elemental composition of the repeating unit (C8H8). The mass spectrum of PS2400 and the expanded view of the isotopic pattern of 23-mer are shown in Fig 3. The observed isotopic pattern of the 23-mer is in very good agreement with the simulated isotopic distribution (R 60,000).

Measurement of synthetic polymers: Polymethyl methacrylate

Polymethyl methacrylate (PMMA) 4000 was measured by using the JMS-S3000 SpiralTOF. The [M+H]+ peaks for PMMA with the basic monomer units of 100u (Fig.1) were observed for this sample. The full PMMA mass spectrum and an expanded view around m/z 4000 are shown in Fig. 2(a) and (b), respectively. The resolving power at m/z 4,000 was approximately 45,000 (FWHM). Also, the mass differences between the 39, 40, and 41- mers had a very good match with the theoretical mass number (100.0524) of the PMMA repeat unit (C5H8O2). A comparison between the 40-mer’s observed and simulated isotopic patterns is shown in Fig. 2(c). The observed isotopic pattern is in very good agreement with the calculated isotopic distribution.

Measurement of synthetic polymers: Polyethylene Glycol

Polyethylene glycol (PEG) 1000 and 8000 were measured by using the JMS-S3000 SpiralTOF. The [M+H]+ peaks for PEG with the basic monomer units of 44u (Fig. 1) were observed for each sample. The mass spectrum for PEG1000 and an expanded view around m/z 1,000 are shown in Fig. 2. The resolving power at m/z 1009 is approximately 60,000 (FWHM). The mass difference between the 21, 22 and 23-mers showed very good agreement with the theoretical mass of the PEG monomer. The full mass spectrum for PEG8000 is shown in Fig. 3(a). The comparison between the observed and simulated isotopic pattern (R 35,000) for the 226mer are shown in Fig. 3(b). The observed isotopic pattern is in very good agreement with the calculated isotopic distribution.

Measurement of a Dendritic MS Reference Standard

A high-quality mass calibration is required to achieve highly accurate mass measurements by mass spectrometry. A polymer or a mixture of peptides is commonly used to calibrate a MALDI-TOF MS system. However, peptides do not necessarily have long-term stability, and a monodisperse polymer does not have a wide m/z range. Sometimes these standards are not well suited for calibration over a wide m/z range. We used a new dendritic MS calibrant (SpheriCal®) to resolve these issues. Here we demonstrate measurement and calibration using the new calibrant with the JEOL SpiralTOF MALDI mass spectrometer.

MALDI-ISD Measurements Using Both the SpiralTOF Mode and the LinearTOF Mode

Matrix assisted laser desorption/ionization (MALDI) combined with in-source decay (ISD) is a useful tool for doing top-down sequencing of intact proteins. In this work, we measured and compared the ISD fragment ions generated for several proteins by using both the high resolution MALDI-Spiral mode and the high sensitivity MALDI-Linear mode available on the JEOL SpiralTOF MALDI-MS system.

MALDI for Small Molecule Analysis: A Complex Drug Mixture

The SpiralTOF’s unique multi-turn ion optics package a very long (17-meter) flight path within a 1-meter space. Electric sectors and Matsuda plates provide perfect focusing to eliminate ion loss due to beam divergence. Post-source decay fragments occurring in the flight path are eliminated by the ion optics, providing a clean and artifact-free background.

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Corona - Glow Discharge (DART Ion Source)

February 22, 2020