DFOcyclo*-p-Phe-NCS was prepared in 5 steps from known precursors. The synthesis and characterization are reported in Supplemental Figures 2–5 (16 (link)–20 (link)). Chelator-to-antibody ratios of 8:1 (p-Bn-SCN-HEHA), 5:1 (DFOcyclo*-p-Phe-NCS), and 4.2:1 (L804-NHS) were reacted in 0.1 M Na2CO3 (pH 9, 37°C for 1 h). L804-ofatumumab was prepared in 0.5 M NH4OAc, pH 5.5, with 1 mM CaCl2. Before 227Th radiolabeling, buffer was exchanged by spin desalting columns (Zeba, 40K, 0.5 mL; Thermo Scientific) to 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (1 M, pH 7). Conjugate ratios were measured by capillary mass spectrometry with an Exactive Plus (Thermo Fisher). Samples were run at a resolving power of 8,750 or 17,500 at 300 m/k and analyzed by Protein Metric Intact.
Exactive plus
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany
The Exactive Plus is a high-resolution accurate-mass (HRAM) mass spectrometer designed for routine analysis of small molecules. It features fast, high-resolution scanning and data acquisition capabilities to support a range of analytical applications.
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Lab products found in correlation
Snap cartridge kp sil columns, by Biotage (1 mentions)
Hp 5 column, by Hewlett-Packard (1 mentions)
Gc 2010 plus, by Shimadzu (1 mentions)
Jnm ecs400, by JEOL (1 mentions)
Synapt g1 hdms, by Waters Corporation (1 mentions)
Thermo orbitrap fusion, by Thermo Fisher Scientific (1 mentions)
P 1010, by Jasco (1 mentions)
Avance 3 500, by Bruker (1 mentions)
Ce 2070, by Jasco (1 mentions)
Nicolet is5 atr, by Thermo Fisher Scientific (1 mentions)
Ultimate 3000 nano, by Thermo Fisher Scientific (1 mentions)
Flash ea 1112, by Thermo Fisher Scientific (1 mentions)
Prominence series, by Shimadzu (1 mentions)
Jnm eca 500 spectrometer, by JEOL (1 mentions)
Jms 700, by JEOL (1 mentions)
P 2200 polarimeter, by Jasco (1 mentions)
Nicolet 6700 spectrometer, by Thermo Fisher Scientific (1 mentions)
Avance 400 mhz spectrometer, by Bruker (1 mentions)
V 630 spectrophotometer, by Jasco (1 mentions)
Triple tof 4600, by AB Sciex (1 mentions)
22 protocols using exactive plus
1
Synthesis and Characterization of Radiolabeled Antibody Conjugates
2
Ester Dance Reaction Protocol
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All reactants or reagents including dry solvents were obtained from commercial suppliers and used as received. All ester dance reactions were performed in 20-ml glass vessel tubes equipped with J. Young O-ring tap and heated (IKA Plate RCT digital) in an oil bath unless otherwise noted. All work-up and purification procedures were carried out with reagent-grade solvents in air. Synthetic methods of all of starting materials are described in the data in the Supplementary Materials.
Analytical thin-layer chromatography (TLC) was performed using Silicagel 70 TLC Plate-Wako (0.25 mm). The developed chromatogram was analyzed by ultraviolet lamp (254 nm). Flash column chromatography was performed with Biotage Isolera equipped with Biotage SNAP Cartridge KP-Sil columns. Preparative TLC (PTLC) was performed using Wakogel B5-F silica-coated plates (0.75 mm) prepared in our laboratory. Gas chromatography (GC) analysis was conducted on a Shimadzu GC-2010 Plus instrument equipped with an HP-5 column (30 m by 0.25 mm, Hewlett-Packard) with n-decane as an internal standard. High-resolution mass spectra (HRMS) were conducted on Thermo Fisher Scientific Exactive Plus (electrospray ionization and direct analysis in real time). Nuclear magnetic resonance (NMR) spectra were recorded on a JEOL JNM-ECS-400 (1H, 400 MHz; 13C, 101 MHz) spectrometer.
Analytical thin-layer chromatography (TLC) was performed using Silicagel 70 TLC Plate-Wako (0.25 mm). The developed chromatogram was analyzed by ultraviolet lamp (254 nm). Flash column chromatography was performed with Biotage Isolera equipped with Biotage SNAP Cartridge KP-Sil columns. Preparative TLC (PTLC) was performed using Wakogel B5-F silica-coated plates (0.75 mm) prepared in our laboratory. Gas chromatography (GC) analysis was conducted on a Shimadzu GC-2010 Plus instrument equipped with an HP-5 column (30 m by 0.25 mm, Hewlett-Packard) with n-decane as an internal standard. High-resolution mass spectra (HRMS) were conducted on Thermo Fisher Scientific Exactive Plus (electrospray ionization and direct analysis in real time). Nuclear magnetic resonance (NMR) spectra were recorded on a JEOL JNM-ECS-400 (1H, 400 MHz; 13C, 101 MHz) spectrometer.
3
Native and Denatured Mass Spectrometry
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In short, native MS was performed on a Synapt G1 HDMS instrument (Waters Corporation) equipped with a 32k RF generator or on a Thermo Scientific Exactive Plus extended mass range (EMR) with a rear-entry ion source (REIS)49 (link). IM data were processed using PULSAR55 (link). Mole fraction was determined from deconvolution of mass spectra with UniDec57 (link). Denatured MS was performed on the front end of the REIS-orbitrap. Tryptic digest analysis and protein sequencing was performed on Thermo Orbitrap Fusion. Further details can be found in Supplementary Methods .
4
Comprehensive Analytical Characterization
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The HR-ESI–MS spectra were measured using an Exactive Plus mass spectrometer (Thermo Fisher Scientific, Bremen, Germany). The 1H-NMR and 13C-NMR spectra were measured using a Bruker Avance III 500 spectrometer (Bruker Daltonics, Bremen, Germany) with TMS as an internal standard. Optical rotations were measured using a JASCO P-1010 spectropolarimeter (Jasco Co., Tokyo, Japan).
5
Optimized Mass Spectrometry for High Mass Proteins
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The Exactive Plus mass spectrometer
(Thermo Fisher Scientific, Bremen, Germany) was modified to include
an adjustable gas supply for the HCD cell, and analogue filters were
removed from the image current preamplifier to allow detection over
the entire frequency range. The control software of the instrument
was modified to allow the standard mass range of this instrument to
be increased from m/z 50–6000
to m/z 400–40 000.
In addition, maximum RF voltages were applied to all RF multipoles
including the C-trap. Instead of trapping in the C-trap, ions were
allowed to enter the HCD cell and were stored there prior to their
return back into the C-trap. Manual tuning of the voltage offset on
the transport octapole was used for mass filtering of the incoming
protein ions, as previously described.23 (link) Frequency reduction on RF multipoles was implemented by adding high-voltage
capacitors to corresponding RF coils and electronic boards automatically
adjusted resonance frequency while keeping the amplitude constant.
(Thermo Fisher Scientific, Bremen, Germany) was modified to include
an adjustable gas supply for the HCD cell, and analogue filters were
removed from the image current preamplifier to allow detection over
the entire frequency range. The control software of the instrument
was modified to allow the standard mass range of this instrument to
be increased from m/z 50–6000
to m/z 400–40 000.
In addition, maximum RF voltages were applied to all RF multipoles
including the C-trap. Instead of trapping in the C-trap, ions were
allowed to enter the HCD cell and were stored there prior to their
return back into the C-trap. Manual tuning of the voltage offset on
the transport octapole was used for mass filtering of the incoming
protein ions, as previously described.23 (link) Frequency reduction on RF multipoles was implemented by adding high-voltage
capacitors to corresponding RF coils and electronic boards automatically
adjusted resonance frequency while keeping the amplitude constant.
6
Comprehensive Analytical Techniques for Compound Characterization
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A capillary liquid chromatographic system consisted of a DiNa S (KYA Technologies, Tokyo, Japan) as a pump, a CE-2070 (JASCO, Tokyo, Japan) as a UV detector, a CHEMINERT (Valco Instruments, Houston, TX) as a sample injector, and a Chemco capillary column conditioner Model 380-b (Chemco, Osaka, Japan) as a column oven. As an HPLC system, a Prominence series (Shimadzu, Kyoto, Japan) was used. FT-IR, NMR, elemental analysis, and fast atom bombardment mass spectrometry (FABMS) were carried out by a Nicolet iS5 ATR (Thermo Fisher Scientific, Yokohama, Japan), a JNM-ECA500 spectrometer (JEOL, Tokyo, Japan), a Flash EA1112 (Thermo Fisher Scientific), and a JMS-700 (JEOL), respectively. LC–MS system was consisted of Ultimate 3,000 nano and Exactive plus (Thermo Fisher Scientific).
7
Orbitrap Mass Spectra Acquisition
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Mass spectra were acquired with an orbitrap mass spectrometer (Exactive Plus; Thermo Fisher Scientific, Waltham, MA). The MS parameters were as follows: capillary temperature = 275 °C, max ion injection time = 10 ms, and 3 microscans for each individual scan.
8
Comprehensive Analytical Characterization
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NMR spectra were recorded on an Avance (400 MHz) spectrometer (Bruker Corporation, Billerica, MA, USA). 1H and 13C NMR chemical shifts were referenced to the CD3OD solvent peaks δH 3.31 and δC 49.15 (Wako, Osaka, Japan). HRESI-MS spectra were measured on an Exactive Plus (Thermo Fisher Scientific Inc., Waltham, MA, USA). ESIMS/MS spectra were measured on a TripleTOF 4600 (AB Sciex Pte. Ltd., Tokyo, Japan) in the positive mode. Optical rotation was determined on a P-2200 polarimeter (JASCO Corporation, Tokyo, Japan) in CH3OH. UV spectra were recorded using a V-630 spectrophotometer (JASCO). IR spectra were measured on a Nicolet6700 spectrometer (Thermo Fisher Scientific Inc.).
9
Zn-PML Interaction with Ru-1 Analyzed
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The mass spectrometry experiments were conducted on an Exactive Plus (Thermo Fisher Scientific, CA, USA) mass spectrometer. Zn-PML was incubated with Ru-1 in 100 mM ammonium acetate buffer (pH = 7.0) at 37 °C for 2 hours. The positive ion mode was used in the ESI-MS experiments. Data were processed using Xcalibur software (version 2.0, Thermo Finnigan).
10
Intracellular Chemical Analysis via InESI-MS
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After extraction of the cytoplasmic chemical constituents from cells, the capillary micropipette was coupled to the InESI device. An AC voltage with an amplitude of 3 kV at approximately 500 Hz was applied to the outside of the spray capillary micropipette. The tip of the micropipette was placed approximately 5 mm away from the orifice of the MS instrument. The MS experiments were performed using an orbitrap mass spectrometer (Exactive Plus, Thermo Fisher Scientific, San Jose, CA, U.S.A.), and tandem mass spectrometry (MS/MS) was conducted with an LTQ mass spectrometer (Thermo Scientific, San Jose, CA, U.S.A.). The MS instrument conditions throughout the experiments were as follows: S lens radio frequency level, 50% (positive mode); capillary temperature, 275 °C; mass resolution, 70 000; microscan, 1; and maximum ion injection time, 10 milliseconds. InESI-MS analysis for all samples were performed in positive mode. The data were acquired via Xcalibur software. The sampling micropipettes were pulled from borosilicate glass capillaries (1.2 mm o.d., 0.9 mm i.d.) by using a P-2000 laser-based micropipette puller (Sutter Instruments, Novato, CA, U.S.A.) with the size of 1–2 μm (i.d.).
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