Exactive plus emr mass spectrometer, by Thermo Fisher Scientific - Product details

1

Native MS Analysis of Antibody-Antigen Complexes

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Native MS was performed as described [54 (link)] with minor modifications. Briefly, stock solutions of STEBVax and IgG-GC132a were buffer-exchanged to 150 mM ammonium acetate (pH 6.8) using Vivaspin 500 centrifugal concentrators (Sartorius, Göttingen, Germany). The working solution was prepared by mixing Ab and antigen solutions with a molar ratio of ~2:1 and a final total protein concentration of ~2 μM. The solution was loaded into NanoES spray capillaries (Thermo Fisher Scientific) on a Nanospray Flex ion source (Thermo Fisher Scientific). Mass spectra were collected on an Exactive Plus EMR mass spectrometer (Thermo Fisher Scientific). The in-source collisional ionization dissociation energy was 20 eV, spray voltage was 1.3 kV, capillary temperature was 100 °C and collision energy was 100 or 200 a.u.

Chen G., Karauzum H., Long H., Carranza D., Holtsberg F.W., Howell K.A., Abaandou L., Zhang B., Jarvik N., Ye W., Liao G.C., Gross M.L., Leung D.W., Amarasinghe G.K., Aman M.J, & Sidhu S.S. (2019). Potent Neutralization of Staphylococcal Enterotoxin B in vivo by antibodies that block binding to the T-cell receptor. Journal of molecular biology, 431(21), 4354-4367.

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2

SFTSV L Protein Mass Spectrometry

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SFTSV L 1–231 aa was buffer exchanged to 150 mM ammonium acetate, pH = 6.8 with the Bio-Spin 6 Columns (Bio-Rad, Hercules, CA). Samples containing 2.5 μM SFTSV L or 2.5 μM SFTSV L plus 100 μM MnCl₂ in the ammonium acetate buffer were loaded into the ES380 borosilicate nanoES spray emitters (Thermo Fisher Scientific, Santa Clara, CA). The emitters were mounted on the Exactive Plus EMR mass spectrometer (Thermo Fisher Scientific, Santa Clara, CA) to acquire mass spectra for 1 to 2 min. Some MS parameters are: 1.5 kV spray voltage, 20 eV in-source CID, 20 HCD cell energy, 100°C transfer capillary temperature. All assays performed at least in triplicate using at least two different protein preparations.

Wang W., Shin W.J., Zhang B., Choi Y., Yoo J.S., Zimmerman M.I., Frederick T.E., Bowman G.R., Gross M.L., Leung D.W., Jung J.U, & Amarasinghe G.K. (2020). The Cap-Snatching SFTSV Endonuclease Domain Is an Antiviral Target. Cell reports, 30(1), 153-163.e5.

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3

Mouse and Human TNF Interaction Analysis

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Mouse and human TNF protein were made up to a concentration of 2 mg/mL (20 mM ammonium acetate, pH 7.4) and desalted and buffer-exchanged using Zeba columns. The protein was then diluted to 20 μM in ammonium acetate buffer. An aliquot of each was mixed 1:1 and analysed at various time points. Human TNF was incubated (1:1, v/v) with compound (20 μΜ), 4% DMSO in 20 mM ammonium acetate) and mixed with mouse TNF (1:1) and tested at various time points using an Advion Nanomate (for sample introduction by nanospray) and ThermoFisher Exactive Plus EMR Mass Spectrometer. The following settings were utilised on the mass spectrometer: scan range 1000 to 8000 m/z; spray voltage 1.5 kV; capillary temperature 150 °C; S-lens RF 200; source DC offset 25 V, injection flatapole 7 V; inter flatapole 6 V; bent flatapole DC 6 V; EMR mode on. Data were processed using ThermoFisher Protein Deconvolution 3.0.

O’Connell J., Porter J., Kroeplien B., Norman T., Rapecki S., Davis R., McMillan D., Arakaki T., Burgin A., Fox III D., Ceska T., Lecomte F., Maloney A., Vugler A., Carrington B., Cossins B.P., Bourne T, & Lawson A. (2019). Small molecules that inhibit TNF signalling by stabilising an asymmetric form of the trimer. Nature Communications, 10, 5795.

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4

SFTSV L Protein Mass Spectrometry

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SFTSV L 1–231 aa was buffer exchanged to 150 mM ammonium acetate, pH = 6.8 with the Bio-Spin 6 Columns (Bio-Rad, Hercules, CA). Samples containing 2.5 μM SFTSV L or 2.5 μM SFTSV L plus 100 μM MnCl₂ in the ammonium acetate buffer were loaded into the ES380 borosilicate nanoES spray emitters (Thermo Fisher Scientific, Santa Clara, CA). The emitters were mounted on the Exactive Plus EMR mass spectrometer (Thermo Fisher Scientific, Santa Clara, CA) to acquire mass spectra for 1 to 2 min. Some MS parameters are: 1.5 kV spray voltage, 20 eV in-source CID, 20 HCD cell energy, 100°C transfer capillary temperature. All assays performed at least in triplicate using at least two different protein preparations.

Wang W., Shin W.J., Zhang B., Choi Y., Yoo J.S., Zimmerman M.I., Frederick T.E., Bowman G.R., Gross M.L., Leung D.W., Jung J.U, & Amarasinghe G.K. (2020). The Cap-Snatching SFTSV Endonuclease Domain Is an Antiviral Target. Cell reports, 30(1), 153-163.e5.

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5

Native Mass Spectrometry of Protein Complexes

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Protein complexes were buffer exchanged in 0.5 M NH₄OAc buffer (pH 6.9) using VIVASPIN 50-kDa MWCO filters (Fisher Scientific, Hanover Park, IL). The final concentration of protein complexes was adjusted to 10 μM. A 10-μl aliquot was loaded into an offline electrospray capillary (Thermo Electron, Madison, WI) to perform the Native-MS measurement on an Exactive Plus EMR mass spectrometer (Thermo Fisher Scientific, Bremen, Germany). The sample solution was infused in positive mode at capillary voltage 1.5–1.8 kV to the instrument with the resolving power set at 17,500. The in-source and HCD collision voltages were adjusted for desolvation of the ions. The instrument was calibrated with the clusters produced by ESI of a CsI solution. The peak picking and data processing was performed with Intact Mass software (Protein Metrics, San Carlos, CA).

Smith M.L., Cui W., Jackobel A.J., Walker-Kopp N, & Knutson B.A. (2018). Reconstitution of RNA Polymerase I Upstream Activating Factor and the Roles of Histones H3 and H4 in Complex Assembly. Journal of molecular biology, 430(5), 641-654.

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6

Native Mass Spectrometry of Protein Complexes

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Protein complexes were buffer exchanged in 0.5 M NH₄OAc buffer (pH 6.9) using VIVASPIN 50-kDa MWCO filters (Fisher Scientific, Hanover Park, IL). The final concentration of protein complexes was adjusted to 10 μM. A 10-μl aliquot was loaded into an offline electrospray capillary (Thermo Electron, Madison, WI) to perform the Native-MS measurement on an Exactive Plus EMR mass spectrometer (Thermo Fisher Scientific, Bremen, Germany). The sample solution was infused in positive mode at capillary voltage 1.5–1.8 kV to the instrument with the resolving power set at 17,500. The in-source and HCD collision voltages were adjusted for desolvation of the ions. The instrument was calibrated with the clusters produced by ESI of a CsI solution. The peak picking and data processing was performed with Intact Mass software (Protein Metrics, San Carlos, CA).

Smith M.L., Cui W., Jackobel A.J., Walker-Kopp N, & Knutson B.A. (2018). Reconstitution of RNA Polymerase I Upstream Activating Factor and the Roles of Histones H3 and H4 in Complex Assembly. Journal of molecular biology, 430(5), 641-654.

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7

Quantitative Intact Protein Mass Spectrometry

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Proteins were resolved by reverse phase separation
with a Vanquish
Flex chromatographic system (Thermo Fisher Scientific) using a MabPac
RP analytical column (Thermo Fisher Scientific) maintained at 50 °C
over an 8 min gradient of Buffer B (47.5% Optima ACN, 47.5% Optima
isopropanol [IPA, Thermo Fisher Scientific], 5% Optima H₂O, and 0.2% FA) from 2 to 100%. Proteins were ionized using a heated
electrospray ionization (HESI) source connected to an Exactive Plus
EMR mass spectrometer (Thermo Fisher Scientific) collecting only intact
protein (MS1) spectra. Quintuplicate injections were performed on
each sample.
Intact mass intensities of all proteoforms were
generated using multiple software packages to demonstrate the comparability
between methods including manual analysis of an averaged scan using
Xtract, sliding window Xtract using BioPharma Finder (Thermo Fisher
Scientific), and deconvolution of an averaged scan using UniDec.^{41 (link)} The percent compound engagement was calculated
for each injection using the following equation
Averages and standard deviations of
the resulting percentages were
generated in Microsoft Excel.

D’Ippolito R.A., Rabara D., Blanco M.A., Alberico E., Drew M.R., Ramakrishnan N., Sontan D., Widmeyer S.R., Scheidemantle G.M., Messing S., Turner D., Arkin M., Maciag A.E., Stephen A.G., Esposito D., McCormick F., Nissley D.V, & DeHart C.J. (2024). A Top-Down Proteomic Assay to Evaluate KRAS4B-Compound Engagement. Analytical Chemistry, 96(13), 5223-5231.

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8

IEX-MS Analysis of Deglycosylated mAbs

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Example 1

Methods

mAbs were subjected to online IEX-MS analysis. An aliquot of the deglycosylated mAb sample (˜50 μg) was injected onto a YMC-BioPro SP-F strong cation exchange (SCX) column (100×4.6 mm) coupled to a Thermo Exactive Plus EMR mass spectrometer or a Thermo Q Exactive plus mass spectrometer for mass measurement. The samples were separated and eluted over a 20 minute pH gradient with ammonium acetate based buffers (buffer A: 20 mM ammonium acetate, pH 5.8; buffer B: 200 mM ammonium acetate, pH 7.6). An analytical splitter (˜200:1 ratio) was connected after the SCX column to reduce the analytical flow to ˜2 μL/min prior to the mass spectrometer for mass detection. The high flow from the splitter was diverted to a Waters ACQUITY photodiode array (PDA) detector for simultaneous UV detection (280 nm).

Results

As a result, an acidic shoulder peak was detected and attributed to a variant of the antibody with a mass increase of approximately 176 Da. However, the mass measurement at the intact level was not accurate because of complications from a glycation modification which elutes in the same acidic shoulder peak and is close in mass (162 Da).

US11366123B2. Glucuronylation as a new acidic post-translational modification on therapeutic monoclonal antibodies (2022-06-21). Regeneron Pharmaceuticals, Inc. [US]. Inventors: Shunhai Wang [US].

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9

Near-native SEC-UV-MS Analysis of mAbs

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Near-native
SEC-UV-MS was carried out using an ACQUITY UPLC Protein BEH SEC column
(4.6 × 300 mm, 1.7 μmm particle size; Waters Corp.). An
isocratic elution using 100 mM CH³COONH⁴, pH
6.0 at 0.25 mL/min, was used for chromatographic separation with a
Vanquish Horizon UHPLC system (Thermo Scientific) equipped with UV
detection at 280 nm. Sample injection amounts of 12.5 μg of
mAb (diluted in 0.15 M sodium phosphate pH 7.0) were used, and data
acquisition was controlled by Chromeleon software (Thermo Scientific).
The outlet of the UHPLC system was directly coupled to an Exactive
Plus EMR mass spectrometer (Thermo Scientific). Data analysis and
deconvolution was completed using Intact Mass from BYOLOGIC PMI (Protein
Metrics Inc.).

Grunert I., Heinrich K., Ernst J., Hingar M., Briguet A., Leiss M., Wuhrer M., Reusch D, & Bulau P. (2022). Detailed Analytical Characterization of a Bispecific IgG1 CrossMab Antibody of the Knob-into-Hole Format Applying Various Stress Conditions Revealed Pronounced Stability. ACS Omega, 7(4), 3671-3679.

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10

Native Mass Spectrometry of Protein Samples

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Protein samples (^~0.5–1 μM) were buffer exchanged into 150 mM ammonium acetate, pH 7.5 using MicroBioSpin6 columns (Bio-Rad 7326221). Native mass spectrometry experiments were carried out on an Exactive Plus EMR mass spectrometer (Thermo Fisher Scientific), which was calibrated in the extended mass range with 5 mg/mL CsI prepared in water. Samples were sprayed from borosilicate capillaries (NanoES spray capillaries, borosilicate; Thermo Fisher Scientific) using a flow rate of 5–40 μL/min, and analyzed in positive ion mode. Experimental parameters were optimized for each run but were generally: spray voltages, 0.8–1.5 kV; injection flatapole, 5; interflatapole lens, 5; bent flatapole, 5; transfer multipole, +4 to −4; C-trap entrance lens, −10 to +10; source DC offset, 25 V; fragmentation collision energy, 20–150 and collision-induced dissociation, 5–150; injection times, 50–200 μs; trapping gas pressure, 7.5; resolution, 17,500 arbitrary units; mass range, 500–20,000 m/z; capillary temperature, 250 °C; S-lens RF value was set to 200 V; microscan s, 10; and automatic gain control was set to 1e⁶.

Kim H., Yen L., Wongpalee S.P., Kirshner J.A., Mehta N., Xue Y., Johnston J.B., Burlingame A.L., Kim J.K., Loparo J.J, & Jacobsen S.E. (2019). The gene silencing protein MORC-1 topologically entraps DNA and forms multimeric assemblies to cause DNA compaction.. Molecular cell, 75(4), 700-710.e6.

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Exactive plus emr mass spectrometer

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10 protocols using exactive plus emr mass spectrometer

Native MS Analysis of Antibody-Antigen Complexes

SFTSV L Protein Mass Spectrometry

Mouse and Human TNF Interaction Analysis

SFTSV L Protein Mass Spectrometry

Native Mass Spectrometry of Protein Complexes

Native Mass Spectrometry of Protein Complexes

Quantitative Intact Protein Mass Spectrometry

IEX-MS Analysis of Deglycosylated mAbs

Near-native SEC-UV-MS Analysis of mAbs

Native Mass Spectrometry of Protein Samples

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