Mass spectrometric analyses of AFCs were performed on a Bruker maXis mass spectrometer coupled to a Dionex Ultimate 3000 RSLC system (Dionex, Germering, Germany). Prior to mass spectrometry (MS) analysis, samples (ca. 5 µg) were desalted on a MassPREP (Waters, Saint-Quentin-en-Yvelines, France) desalting cartridge (2.1 × 10 mm, Waters) heated at 80 °C using 0.1% formic acid as solvent A and 0.1% formic acid in acetonitrile as solvent B at 500 µL/min. After 1 min, a linear gradient from 5 to 90% B in 1.5 min was applied; the first 1.5 min were diverted to waste. MS data were acquired in positive mode with an ESI source over the m/z range from 900 up to 5000 at 1 Hz and processed using DataAnalysis 4.4 software (Bruker, Bremen, Germany) and the MaxEnt algorithm for spectral deconvolution.
Massprep
Manufactured by Waters Corporation
Sourced in United States
The MassPREP is a sample preparation system designed for high-throughput protein analysis. It automates the process of protein purification and digestion, allowing for faster and more consistent sample preparation prior to mass spectrometry analysis.
Automatically generated - may contain errors
Lab products found in correlation
Maxis mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Dataanalysis 4, by Bruker (1 mentions)
Dionex ultimate 3000 rslc system, by Thermo Fisher Scientific (1 mentions)
Vion ims qtof, by Waters Corporation (1 mentions)
Synapt g2 quadrupole time, by Waters Corporation (1 mentions)
C4 column, by Waters Corporation (1 mentions)
Sequencing grade modified porcine trypsin, by Promega (1 mentions)
Bradford assay, by Bio-Rad (1 mentions)
7 protocols using massprep
1
Mass Spectrometric Analysis of AFCs
2
Mass Spectrometry Analysis of Bispecific Nanobody
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The purified bispecific nanobody (1 mg/mL) was concentrated in a 0.05 mmol/L NH4HCO3 solution. An ultrahigh pressure liquid system (ACQUITY I-Class) was connected to a VION IMS-Q-TOF (Waters, Massachusetts, USA). The MassPREP (Waters, Massachusetts, USA) desalting column was used for UPLC with a controlled column temperature of 60 ℃. Mobile phase A was a 0.1% formic acid-water solution, and mobile phase B was a 0.1% formic acid-acetonitrile solution. The system was preequilibrated with 95% mobile phase A, and then 0.5 µg of the bispecific nanobody was injected and eluted with a gradient at a flow rate of 0.2 mL/min. The mass spectrometry conditions were as follows: capillary voltage, 2.0 kV; sampling cone voltage, 60 V; ion source temperature, 115 °C; desolvent gas temperature, 500 °C; and desolvent gas flow rate, 900 L/min. Data were collected by UNIFI 1.8, and mass (Da)-intensity was obtained by deconvolution analysis.
3
Quantitative Peptide Standard Preparation
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Predigested peptide standards
(MassPREP) were
purchased from Waters (Milford, MA, USA). Peptide standards (SDGRG
and GRGDS) were purchased from Sigma-Aldrich. All peptide standards
were received as a lyophilized powder and reconstituted in 10 mM ammonium
acetate in water to a final concentration of 10 μg/mL. The MassPREP
digestion standard mix contained approximately equimolar concentrations
of four tryptically digested proteins: Alcohol Dehydrogenase (ADH,
yeast), Serum Albumin (BSA, bovine), Phosphorylase B (PHOSPH, Rabbit)
and Enolase (ENOLASE, yeast). Peptide identifications were assigned
on the basis of exact mass of all possible tryptic peptides (no missed
cleavages) produced by the Expert Protein Analysis System (ExPASy)
PeptideMass proteomics tool25 (link) (Swiss Institute
of Bioinformatics, Lausanne, Switzerland) using the SWISS-PROT database
entry number for each intact protein (P00330, P02769, P00924, and P00489, respectively). A full list of
identified peptides can be found in theSupporting
Information .
(MassPREP) were
purchased from Waters (Milford, MA, USA). Peptide standards (SDGRG
and GRGDS) were purchased from Sigma-Aldrich. All peptide standards
were received as a lyophilized powder and reconstituted in 10 mM ammonium
acetate in water to a final concentration of 10 μg/mL. The MassPREP
digestion standard mix contained approximately equimolar concentrations
of four tryptically digested proteins: Alcohol Dehydrogenase (ADH,
yeast), Serum Albumin (BSA, bovine), Phosphorylase B (PHOSPH, Rabbit)
and Enolase (ENOLASE, yeast). Peptide identifications were assigned
on the basis of exact mass of all possible tryptic peptides (no missed
cleavages) produced by the Expert Protein Analysis System (ExPASy)
PeptideMass proteomics tool25 (link) (Swiss Institute
of Bioinformatics, Lausanne, Switzerland) using the SWISS-PROT database
entry number for each intact protein (P00330, P02769, P00924, and P00489, respectively). A full list of
identified peptides can be found in the
Information
4
Mass Spectrometry Analysis of Proteins
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Sample solution was diluted to a final concentration of 1 mg/mL in 0.05 M Tris-HCl (pH 7.5). Analysis of intact protein and reduced protein was run on a MassPrep™ micro desalting column (Waters, USA) with a flow rate of 0.2 mL/min. The gradient began at 10% phase B for 1 min, and a linear gradient from 10% phase B to 60% phase B in 10 min was then performed, followed by an increase to 90% phase B in 2 min, followed by holding at 90% for another 5 min. The Q-TOF mass spectrometer was run in positive ion mode. Sample cone voltage, capillary voltage, mass resolution, and m/z range were set at 60 V, 3000 V, 30000, and 250–4000, respectively. The mass spectrometer was calibrated with NaI solution at a final concentration of 2 μg/μL in water/isopropanol (50/50) and was tuned with leucine enkephalin solution (LE, 1 ng/μL) in water/acetonitrile (50/50) with 0.1% formic acid. The mass correction was conducted using ion of LE at m/z 556.2771. The raw spectra data acquired were then deconvoluted and analyzed using BiopharmaLynx software (version 1.3.3 Build 7).
5
Characterizing Monoclonal Antibody Glycosylation
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Molecular masses of CMAB008 and the reference product were determined with a Waters Synapt G2 quadrupole time-of-flight mass spectrometry (MS) system. N-linked polysaccharides of Abs were deglycosylated by adding PNGase F. Proteins were reduced with DTT. Intact and reduced samples were analyzed by reverse-phase liquid chromatography-MS. Reverse-phase desalting separations of intact samples were performed on a Waters MassPrep micro-desalting column (2.1×5.0 mm) using a gradient (3–9 minutes, 5%–90% B). Reverse-phase separations of reduced samples were performed on a Waters C4 column (2.1×50 mm, 1.7 µm) using a gradient (6–18 minutes, 5%–45% B). The mobile phase B was acetonitrile containing 0.1% formic acid, whereas mobile phase A was water containing 0.1% formic acid. The flow rate was maintained at 0.40 mL/minute and column temperature at 80°C to the desalting column and 40°C to the C4 column, respectively. MS analysis was performed on the Synap G2 system in positive-ion mode. Desolvation gas and source temperatures were set to 350°C and 120°C, respectively. Capillary and cone voltages were set at 3,000 and 45 V, respectively. Transfer-collision energy was set at 6.0 V. The scan range was set to 500–3,000 m/z. Raw protein spectra of intact and reduced samples were deconvoluted before evaluation using the maximum-entropy algorithm in BiopharmaLynx MS software.
6
Proteome Analysis of TBEC Samples
7
Protein Extraction and Tryptic Digestion
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Cells were lysed and protein concentration was determined using the Bradford assay (Bio-Rad). Protein samples (10 µg) were reduced in Laemmli buffer for 5 min at 95 °C and then loaded on a homemade one-dimensional SDS-PAGE gel (8% separating gel overlaid with a 5% stacking gel). Electrophoresis was stopped as soon as the protein sample entered the separating gel. The gel was fixed, stained with Coomassie Blue R250, and a single band containing the whole sample was excised from the gel, placed in 96-well plate, and then washed with water. Tryptic digestion was performed on a MassPrep liquid handling robot (Waters, Milford, MA, USA) according to the manufacturer’s specifications and to the protocol of Shevchenko et al [55 (link)] with the modifications suggested by Havlis et al [56 (link)]. Briefly, proteins were reduced with 10 mM DTT and alkylated with 55 mM iodoacetamide. Trypsin digestion was performed using 126 nM of modified porcine trypsin (sequencing grade, Promega, Madison, WI, USA) at 37 °C overnight. Digestion products were extracted using 1% formic acid, 2% acetonitrile followed by 1% formic acid, and 50% acetonitrile. The recovered extracts were pooled, vacuum centrifuge dried, and then resuspended at 0.4 µg/µL with 2% acetonitrile, 0.05% trifluoroacetic acid.
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