All MS analyses were conducted on a Q Exactive HF instrument (Thermo Fisher Scientific) in positive mode. Chromatography was accomplished using a nanoAcquity UPLC, equipped with a nanoAcquity NPLC Symmetry C18 trap column, and an Acquity UPLC Peptide BEH C18 analytical column (Waters), heated to 37 °C, and a Triversa Nanomate source (Advion). We used 1% acetonitrile/0.1% formic acid for mobile phase A and 99% acetonitrile/0.1% formic acid for mobile phase B. We trapped analytes at 4 μl/min for 4 min in mobile phase A. Separation was done at 0.5 μl/min flow rate using the following gradient: 2 to 5% mobile phase B over 0 to 3 min, 5 to 40% B over 3 to 93 min, 40% B over 93 to 98 min, 40 to 98% B over 98 to 100 min, 98% B over 100 to 105 min, 98 to 2% B over 105 to 106 min, and 2% B over 106 to 120 min.
Triversa nanomate source
Manufactured by Advion
Sourced in United States
The Triversa Nanomate source is a lab equipment product designed for nano-electrospray ionization. It provides a reliable and accurate method for introducing samples into mass spectrometers. The Triversa Nanomate source is a key component for various analytical applications requiring high-sensitivity mass spectrometric analysis.
Automatically generated - may contain errors
Lab products found in correlation
Acquity uplc peptide beh c18 analytical column, by Waters Corporation (2 mentions)
Nanoacquity nplc symmetry c18 trap column, by Waters Corporation (2 mentions)
Q exactive hf instrument, by Thermo Fisher Scientific (1 mentions)
Nanoacquity uplc, by Waters Corporation (1 mentions)
Sephadex g 25 column, by GE Healthcare (1 mentions)
Q exactive hf mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Q exactive hf mass spectrometer, by Waters Corporation (1 mentions)
Ltq orbitrap velos etd, by Thermo Fisher Scientific (1 mentions)
4 protocols using triversa nanomate source
1
Nanoflow LC-MS/MS for Protein Quantification
2
Determining Light Chain Mispairing in Multispecific Antibodies
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Example 1
Method for the Determination of Light Chain Mispairing of a Multispecific Antibody after Limited Proteolytic Digestion
The expected primary structures were analyzed by electrospray ionization mass spectrometry (ESI-MS) of the limited LysC digested CrossMabs. Advantageously the antibody has been deglycosylated in advance.
The VH/VL CrossMabs was deglycosylated with N-Glycosidase F in a phosphate or Tris or histidine buffer at 37° C. for up to 17 h at a protein concentration of 1 mg/mL and an antibody: enzyme ratio of 100:1. The limited Lys-C (Roche Diagnostics GmbH, Mannheim, Germany) digestions was performed with 100 μg deglycosylated VH/VL CrossMabs in a Tris buffer pH 8 at 37° C. for 40 min.
Prior to mass spectrometry the samples were desalted via HPLC on a Sephadex G25 column (GE Healthcare).
The total mass was determined via ESI-MS on a maXis 4G UHR-QTOF MS system (Bruker Daltonik) equipped with a TriVersa NanoMate source (Advion).
3
Glycopeptide Characterization by Mass Spectrometry
4
Online LC-MS/MS Peptide Analysis
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Online LC‐MS/MS was performed on a Dionex UltiMate 3000 RLSCnano (Thermo Fisher Scientific, Bremen, Germany) system coupled to an LTQ‐Orbitrap Velos ETD (Thermo Fisher Scientific). Peptides were loaded onto a 150 mm Acclaim PepMap100 C18 column (LC Packings, Sunnyvale, CA, USA) in formic acid (0.1% (v/v)), and separated over a 90 min. linear gradient from 3.2% to 44% mobile phase B (acetonitrile with formic acid (0.1% (v/v)) with a flow rate of 350 nl/min. The column was then washed with 90% mobile phase B before re‐equilibrating at 3.2% mobile phase B. The column was maintained at 35°C. The LC system was coupled to an Advion Biosciences TriVersa NanoMate source (Ithaca, NY, USA) which infused the peptides with a spray voltage of 1.7 kV. Peptides were infused directly into the mass spectrometer. The mass spectrometer performed a full FT‐MS scan (m/z 380–1600) and subsequent collision‐induced dissociation (CID, 35% normalized collision energy NCE) MS/MS scans of the three most abundant ions followed by higher energy collisional dissociation (HCD 55 NCE) of the same three ions. Analysed ions were placed on an exclusion list for 60 s. The CID and HCD spectra were used for peptide identification and quantification respectively. Each SCX set (i.e. the four SCX fractions from each sample) was run in sequence followed by a blank and repeated in triplicate.
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