Mass spectroscopic analysis of the PEGylated isomers was carried out using a JEOL AccuTOF™ time-of-flight mass spectrometer (JEOL Ltd., Tokyo, Japan). The AccuTOF analyzer performs mass measurement of compounds of high molecular weight using ion optics and an ADC-based digitizer to deliver a dynamic range exceeding 4 orders of magnitude. The mass spectrometer was equipped with orthogonal spray electrospray ionization (ESI) ion source. Before analysis, the PEGylated isomers were dissolved in HPLC grade methanol. A 50 μL sample was then injected through a Rheodyne 6-port valve injector. The mass spectrometer was operated in positive-ion mode (ESI + ve) with an applied needle voltage of 2000 V. The atmospheric pressure interface potential was set to the following values: orifice 1 = 55 V, ring lens voltage = 5 V, and orifice 2 = 6 V. The detector voltage was set to 1900 V. Orifice 1 temperature was adjusted to 80 °C with dissolving temperature at 250 °C. Nebulizing gas flow was set at 2 L/min rate whereas the desolvation gas (N2) flow was adjusted to 5 L/min rate.
Accutof
Manufactured by JEOL
Sourced in Japan, United States
The AccuTOF is a high-resolution time-of-flight mass spectrometer (TOFMS) designed for accurate mass measurements. It utilizes a unique ion optics system to deliver high mass resolving power and mass accuracy for a wide range of applications.
Automatically generated - may contain errors
Lab products found in correlation
Discover system, by CEM Corporation (2 mentions)
Ascend 400, by Bruker (2 mentions)
1100 hplc system, by Agilent Technologies (2 mentions)
Inova 400 mhz nmr spectrometer, by Agilent Technologies (2 mentions)
Avance dmx 500, by Bruker (1 mentions)
Tensor 27 spectrophotometer, by Bruker (1 mentions)
Microflex lrf maldi tof, by Bruker (1 mentions)
Irdye700dx, by LI COR (1 mentions)
C18 reversed phase column, by Phenomenex (1 mentions)
High performance liquid chromatography (hplc), by Phenomenex (1 mentions)
Amicon ultra 0.5 spin filter units, by Merck Group (1 mentions)
Ecs 400, by JEOL (1 mentions)
Silica gel 60n, by Kanto Chemical (1 mentions)
Wakogel c 300, by Fujifilm (1 mentions)
Silica gel 60 f254 1, by Merck Group (1 mentions)
Ft ir 4100, by Jasco (1 mentions)
Avance dmx400, by Bruker (1 mentions)
Xterra rp18, by Agilent Technologies (1 mentions)
Agilent 1100 hplc system, by Agilent Technologies (1 mentions)
20 protocols using accutof
1
Mass Spectrometric Analysis of PEGylated Isomers
2
DART-MS Analysis of Indigoidine in Vibrio fischeri Colonies
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Direct analysis in real time-mass spectrometry (DART-MS) analysis was performed using a JEOL AccuTOF with DART ion source (IonSense, Inc., Saugus, MA, USA). High purity helium 5.0–6.0 grade (greater than 99.999% purity) was heated to 300°C and used for ionization. Five locations were selected on JC1 colonies in the presence or absence of V. fischeri, including (A) center of colony, (B) midpoint between center and edge of colony, (C) edge of colony, (D) ZOI (in the absence of V. fischeri sample was obtained from a point equidistant from colony edge), and (E) outside ZOI. At each location a sterile single use syringe needle (BD Medical, Franklin Lakes, NJ, USA) was placed in the sample and then placed between the DART ion source and the MS inlet. Positive ion MS data were obtained over a m/z range of 60–700 and relative percent abundance was obtained for the indigoidine ion. Standards were run after sampling each colony and mass spectral data were monitored in real time to ensure no residual indigoidine remained after each sample. DART-MS is only semi-quantitative due to the potential for differential ionization, suppression of ions, and/or changes in sample concentration in the DART ion source (Sanchez et al., 2011 (link)). Therefore, relative indigoidine ion abundance was used to generate heatmaps representing a gradient from less abundance (black) to more abundance (red).
3
Mass Spectrometry Protocol for Compound Analysis
4
Characterization of Organic Compounds
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All reagents were obtained from commercial suppliers and used without further purification. Merck silica gel (type 60, 0.063–0.200 mm) was used for column chromatography. All compounds were characterized by IR spectra, recorded on an FT-IR Bruker Tensor 27 spectrophotometer, by means of attenuated total reflection (ATR) technique, and all data is expressed as wavenumbers (cm−1). Melting points were obtained on a Fisher–Johns apparatus and are uncorrected. NMR spectra were recorded on a Bruker Ascend-400 (400 MHz) and Bruker Avance DMX-500 (500 MHz) spectrometers in CDCl3 or acetone-d6 and chemical shifts are given in ppm with tetramethylsilane (TMS) as the reference. Direct analysis in real time mass spectrometry (DART-MS) were obtained on a Jeol AccuTOF; the values of the signals are expressed as mass/charge units (m/z). Microwave irradiation experiments were performed using a Discover System (CEM Corporation, Matthews, NC, USA) single-mode microwave with standard sealed microwave glass vials. The reaction temperature was monitored by an IR sensor on the outside wall of the reaction.
5
Flavylium Cation and trans-Chalcone Analysis
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Purified samples of both the flavylium cation and the trans-chalcone of 1 were analysed by high-resolution electron spray ionisation mass spectrometry (HR-ESI-MS). The spectra were recorded using a JEOL AccuTOF instrument (JEOL USA, Inc., Peabody, MA, USA). See supplementary Figures S7 and S8 for details about the individual MS spectra.
6
Flame-based Mass Spectrometry for Diamond Synthesis
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Positive ions in the C2H4/C2H2/O2 flame were detected using a time-of-flight mass spectrometer (MS, AccuTOF™, JEOL USA, Inc.), as shown in Figure S2 in the Supplementary Information . A stainless steel orifice with an inner diameter of 400 μm on the mass spectrometer collects ions in open air. The combustion torch was fixed on a motorized X-Y-Z stage and the relative position of the flame to the orifice was precisely adjusted so that the tip of the flame inner cone was right at front of the orifice under all excitation conditions. The flame-to-orifice distance for the MS detection was the same as the flame-to-substrate distance for diamond deposition. In this way, the detected species are believed to be the same as what exist in the diamond growth. The MS data were analyzed using the TSSPro software (Shrader Analytical and Consulting Laboratories, Inc. Version 3.0) and the MS Tools software (JEOL USA, Inc.).
7
Conjugation of Val-Cit-PABA-β-alaninoyl-maytansinoid
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Example 4
To a suspension of Val-Cit-PABA-β-alaninoyl-maytansinoid (commercially available from Concortis, San Diego, USA) (27 mg, 0.022 mmol) in MeCN (2 mL) was added triethylamine (9.2 μL, 6.7 mg, 0.066 mmol) and a solution of BCN-PEG2-C(O)OSu carbonate 35 (9.2 mg, 0.022 mmol) in MeCN (1 mL). After 23 h, the mixture was poured out in a mixture of EtOAc (20 mL) and water (20 mL). After separation, the organic phase was dried (Na2SO4) and concentrated. After purification via column chromatography (EtOAc→MeOH/EtOAc 1/4) 22 mg (0.015 mmol, 70%) of the desired product was obtained. LRMS (ESI+) calcd for C70H97ClN10O20 (M+H+) 1432.66, found 1434.64.
Conjugations
General Protocol for Mass Spectral Analysis of IgG
A solution of 50 μg (modified) IgG, 1 M Tris-HCl pH 8.0, 1 mM EDTA and 30 mM DTT with a total volume of approximately 70 μL was incubated for 20 minutes at 37° C. to reduce the disulfide bridges allowing to analyze both light and heavy chain. If present, azide-functionalities are reduced to amines under these conditions. Reduced samples were washed trice with milliQ using an Amicon Ultra-0.5, Ultracel-10 Membrane (Millipore) and concentrated to 10 μM (modified) IgG. The reduced IgG was analyzed by electrospray ionization time-of-flight (ESI-TOF) on a JEOL AccuTOF. Deconvoluted spectra were obtained using Magtran software.
8
NMR Characterization of Organic Compounds
9
Synthesis and Characterization of FX12
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FX12 was synthesized using methyl benzo[b]thiophene-2-carboxylate as the starting material. Briefly, a solution of methyl benzo[b]thiophene-2-carboxylate (192 mg, 1 mmol) in dichloromethane (10 ml) was added 3-chloroperbenzoic acid (m-CPBA, 2 mmol) in portions over 20 min. The reaction was stirred at 50°C for 5 h and then cooled to room temperature. To the reaction mixture was added 0.5 M sodium hydroxide (20 ml). The solution was stirred for another 15 min. The aqueous layer was removed. The organic phase was washed using brine, dried over Na2SO4, and concentrated. The crude material was purified by flash chromatography to give compound FX12: 1H NMR (Varian INOVA 400 MHz, CDCl3): δ 7.99 (d, J = 0.8 Hz, 1H), 7.78 (d, J = 8.0 Hz, 1H), 7.71-7.63 (m, 2H), 7.53 (dd, J = 1.6 Hz, J = 7.2 Hz, 1H) and 13C NMR (Varian INOVA 100 MHz, CDCl3): δ 154.4, 140.3, 134.9, 134.1, 133.4, 127.4, 127.2, 122.2, 53.4; and HRMS (ESI, JEOL AccuTOF with ESI/APCI ion sources coupled to an Agilent 1100 HPLC system): The exact mass calculated for C10H9O4S [M+H]+ 225.0222, found 225.0224.
10
Fc Fragmentation of Modified IgG
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Prior to mass spectral analysis, (modified) IgG
was treated with IdeS (Fabricator) for analysis of the Fc/2 fragment.
A solution of 10 μg (modified) IgG was incubated for 1 h at
37 °C with 0.5 μL of IdeS (50 U/μL) in PBS pH 7.4.
Samples were diluted by MQ to 40 μL followed by electrospray
ionization time-of-flight (ESI-TOF) analysis on a JEOL AccuTOF. Deconvoluted
spectra were obtained using Magtran software.
was treated with IdeS (Fabricator) for analysis of the Fc/2 fragment.
A solution of 10 μg (modified) IgG was incubated for 1 h at
37 °C with 0.5 μL of IdeS (50 U/μL) in PBS pH 7.4.
Samples were diluted by MQ to 40 μL followed by electrospray
ionization time-of-flight (ESI-TOF) analysis on a JEOL AccuTOF. Deconvoluted
spectra were obtained using Magtran software.
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