N-linked glycans were enzymatically cleaved from SDS-PAGE gels using PNGaseF prepared in-house. Released N-glycans were fluorescently labelled with 2-aminobenzoic acid (2-AA) for analysis by hydrophilic interaction chromatography ultra-high performance liquid chromatography (HILIC-UHPLC) with a Waters Acquity UPLC instrument, as previously described38 (link). Glycan compositions were determined using traveling wave (TW) IM-MS measurements performed on a Synapt G2Si instrument (Waters, Manchester, UK). For each analysis 2 µl of sample were cleaned with a Nafion 117 membrane and directly infused by nano-electrospray ionization (nano-ESI) from gold-coated borosilicate glass capillaries as previously described16 (link),39 (link). Instrument settings were as follows: capillary voltage, 0.8–1.0 kV; sample cone, 100 V; extraction cone, 25 V; cone gas, 40 l/h; source temperature, 150 °C; trap collision voltage, 4–160 V; transfer collision voltage, 4 V; trap DC bias, 35–65 V; IMS wave velocity, 450 m/s; IMS wave height, 40 V; trap gas flow, 2 ml/min; IMS gas flow, 80 ml/min. Data was acquired and processed with MassLynx v4.1 and Driftscope version 2.8 software (Waters, Manchester, UK).
Driftscope version 2
Manufactured by Waters Corporation
Sourced in United Kingdom
The Driftscope version 2.8 is a high-precision analytical instrument designed for specialized laboratory applications. It provides accurate measurements and analysis capabilities for various sample types. The core function of the Driftscope 2.8 is to facilitate precise and reliable data collection and processing.
Automatically generated - may contain errors
Lab products found in correlation
Masslynx v4, by Waters Corporation (5 mentions)
Synapt g2 si, by Waters Corporation (4 mentions)
Acquity uplc, by Waters Corporation (2 mentions)
Synapt g2 si mass spectrometer, by Waters Corporation (1 mentions)
Masslynx, by Waters Corporation (1 mentions)
Empower software, by Waters Corporation (1 mentions)
Synapt g2si instrument, by Waters Corporation (1 mentions)
Empower 3, by Waters Corporation (1 mentions)
Masslynx version 4, by Waters Corporation (1 mentions)
10 protocols using driftscope version 2
1
Enzymatic Release and Mass Spectrometric Analysis of N-Linked Glycans
2
N-Glycan Analysis by IM-ESI MS
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IM-ESI MS and tandem MS of released N-linked glycans were performed on a Synapt G2Si instrument (Waters) as previously described (44 (link)). Glycans were purified on a Nafion membrane before injection. Data acquisition and processing were carried out using MassLynx v4.11 and Driftscope version 2.8 software (Waters).
3
Quantitative Glycan Characterization by MS
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The quantifications and structural characterization of the total glycan pool was achieved by cleaving the N-inked glycans from the surface of the glycoprotein using an in-gel digestion with peptide N-glycosidase F (PNGaseF). The resultant glycans were separated into two aliquots. The first was derivatized with 2-aminobenzoic acid (2-AA) and subjected to HILIC-UPLC analysis using an Acquity UPLC (Waters). To quantify the oligomannose content of the released glycans, the labeled samples were treated with endoglycosidase H (endoH), which selectively cleaves oligomannose glycans. Data analysis and interpretation were performed using Empower software (Waters). The second aliquot of released glycans was subjected to negative ion electrospray ion mobility mass spectrometry using a Synapt G2Si mass spectrometer (Waters). Glycan compositions were determined using collision induced dissociation (CID) fragmentation. Data analysis was performed using Waters Driftscope (version 2.8) software and MassLynxTM (version 4.1). Spectra were interpreted as described previously (Harvey et al., 2009 (link)). The glycan compositions were used to generate a sample-specific glycan library that was used to search the glycopeptide data to minimize the number of false-positive assignments in site-specific analysis.
4
N-Glycan Analysis by Ion-Mobility MS
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Prior to ion-mobility electrospray ionisation MS and tandem MS analysis, PNGase F released N-linked glycans were purified on a Nafion® 117 membrane (Sigma-Aldrich) and a trace amount of ammonium phosphate was added to promote phosphate adduct formation. Glycans were analyzed by direct infusion using a Synapt G2Si instrument (Waters) with the following settings: capillary voltage, 0.8–1.0 kV; sample cone, 150 V; extraction cone, 150 V; cone gas, 40 l/h; source temperature, 80 °C; trap collision voltage, 4–160 V; transfer collision voltage, 4 V; trap DC bias, 60 V; IMS wave velocity, 450 m/s; IMS wave height, 40 V; trap gas flow, 2 ml/min; IMS gas flow, 80 ml/min. Data were acquired and processed with MassLynx v4.1 and Driftscope version 2.8 software (Waters).
5
Glycan Profiling using IM-MS and Byonic
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The integration of peaks corresponding to fluorescently labeled N-glycans was performed using Empower 3.0 (Waters, Manchester, UK) (Figures 2 and S3 ). The IM-MS data used to generate the glycan library were acquired and processed with MassLynx v4.1 and Driftscope version 2.8 software (Waters, Manchester, UK) (Figure S4 ). Chromatographic areas were extracted for site-specific analysis using Byonic™ (Version 2.7) and Byologic™ software (Version 2.3) by Protein Metrics (Figure 3 , Figure 4 , Figure 5 , S2 , and S5 ).
6
Quantification of Compounds by IM-MS
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Mobilograms and MS spectra were extracted from MassLynx v4.1 and calibration curves were computed in Prism software version 5.01 (GraphPad, La Jolla, CA, USA). Recommendations for reporting results of IM-MS measurements [14 (link)] were followed. As IM was used here as a separation method and not for structural analyses, the drift times (DT) are reported as IM data. Two-dimensional mobility maps (mass-over-charge (m/z) vs. DT maps) were obtained using Driftscope version 2.9 (Waters). The previously described MobA method [9 (link)] was used for data extraction: the mobility peaks of the compounds of interest were first extracted from the regions of the mass spectra specific to each of the targeted compounds (extracted ion mobilograms (XIM)). The obtained XIM were then automatically integrated to retrieve the peak areas using MassLynx software [9 (link),12 (link)], and the normalized responses were calculated using the ratio of SLX mobility peak area to its IS mobility peak area.
7
Ion Mobility-Mass Spectrometry Data Analysis
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Chromatograms and spectra were opened in MassLynx v4.1. Calibration curves were computed in Prism software version 5.01 (GraphPad, LaJolla, CA, USA). In the present study, recently released recommendations for reporting IM–MS measurements [22 (link)] were followed. As IMS is used here as a separative method and not for structural analyses, the DT is reported as IMS data. 2D mobility maps (m/z vs. DT maps) were obtained using Driftscope version 2.9 (Waters).
For IMS data, two methods of data extraction were adopted, MassI and MobA, as previously described [7 (link)]. Briefly, MassI consisted in extracting the maximum intensity of the m/z peaks of interest in the mass spectra combined with the full TIC, whereas MobA consisted in automatically integrating the area of the extracted mobility peaks corresponding to the compounds of interest using MassLynx software [7 (link)].
For IMS data, two methods of data extraction were adopted, MassI and MobA, as previously described [7 (link)]. Briefly, MassI consisted in extracting the maximum intensity of the m/z peaks of interest in the mass spectra combined with the full TIC, whereas MobA consisted in automatically integrating the area of the extracted mobility peaks corresponding to the compounds of interest using MassLynx software [7 (link)].
8
Comparative ECD Analysis of Biomolecules
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This study was designed
to demonstrate the ultimate performance for ECD coupled with cIMS
for continuous infusions, and not LC-MS/MS experiments. For the post-cIMS
ECD of trastuzumab peptides and pre-cIMS ECD of ubiquitin experiments,
1 and 4 min of data were accumulated at an acquisition rate of 2 scans/s,
respectively. Pre-cIMS ECD of carbonic anhydrase experiments utilized
10 min of data averaging. Waters MassLynx version 4.1 was used to
generate mass spectra as a function of drift time and the resulting
centroided mass spectra. Waters Driftscope version 2.9 was used to
produce 2D mobiligrams and extract mass spectra for a portion of the
2D mobiligram. Centroid mass spectra were converted to MGF file type,
and the peak intensity threshold was applied (intensity of 50 or 500).
The MGF files were used as the input for the LCMS Spectator (version
1.1.7023.32278;https://github.com/PNNL-Comp-Mass-Spec/LCMS-Spectator/releases ). ECD spectra were annotated with b/y and c/z ions, and product ions
maps were generated using an LCMS Spectator with 10 ppm mass error
and Pearson correlation of 0.8 for isotopic distributions.
to demonstrate the ultimate performance for ECD coupled with cIMS
for continuous infusions, and not LC-MS/MS experiments. For the post-cIMS
ECD of trastuzumab peptides and pre-cIMS ECD of ubiquitin experiments,
1 and 4 min of data were accumulated at an acquisition rate of 2 scans/s,
respectively. Pre-cIMS ECD of carbonic anhydrase experiments utilized
10 min of data averaging. Waters MassLynx version 4.1 was used to
generate mass spectra as a function of drift time and the resulting
centroided mass spectra. Waters Driftscope version 2.9 was used to
produce 2D mobiligrams and extract mass spectra for a portion of the
2D mobiligram. Centroid mass spectra were converted to MGF file type,
and the peak intensity threshold was applied (intensity of 50 or 500).
The MGF files were used as the input for the LCMS Spectator (version
1.1.7023.32278;
maps were generated using an LCMS Spectator with 10 ppm mass error
and Pearson correlation of 0.8 for isotopic distributions.
9
Glycan Profiling of BG505 SOSIP.664 by IM-MS
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To guide subsequent glycopeptide analyses, we performed IM-MS on a separate, unlabeled aliquot of PNGase F-released glycans from the BG505 SOSIP.664 protein. Glycan compositions were determined using traveling wave IM-MS measurements performed on a Synapt G2Si instrument (Waters, Manchester, UK). The glycan sample was cleaned with a Nafion 117 membrane and a trace amount of ammonium phosphate was added to promote phosphate adduct formation. Glycans were analyzed by nano-electrospray with direct infusion with the following settings: capillary voltage, 0.8–1.0 kV; sample cone, 100 V; extraction cone, 25 V; cone gas, 40 L/h; source temperature, 150°C; trap collision voltage, 4–160 V; transfer collision voltage, 4 V; trap direct current bias, 35–65 V; IMS wave velocity, 450 m/s; IMS wave height, 40 V; trap gas flow, 2 mL/min; IMS gas flow, 80 mL/min. Data were acquired and processed with MassLynx v4.1 and Driftscope version 2.8 software (Waters, Manchester, UK). Structural assignments were based on previously described IM-MS of BG505 SOSIP.664 glycans (Behrens et al., 2016 (link)).
10
IM-MS Glycan Characterization of BG505 SOSIP.664
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To guide subsequent glycopeptide analyses, we performed IM-MS on a separate, unlabeled aliquot of PNGase F-released glycans from the BG505 SOSIP.664 protein. Glycan compositions were determined using traveling wave IM-MS measurements performed on a Synapt G2Si instrument (Waters, Manchester, UK). The glycan sample was cleaned with a Nafion 117 membrane and a trace amount of ammonium phosphate was added to promote phosphate adduct formation. Glycans were analyzed by nano-electrospray with direct infusion using a Synapt G2Si instrument (Waters, Manchester, UK) with the following settings: capillary voltage, 0.8-1.0 kV; sample cone, 100 V; extraction cone, 25 V; cone gas, 40 L/h; source temperature, 150 °C; trap collision voltage, 4-160 V; transfer collision voltage, 4 V; trap direct current bias, 35-65 V; IMS wave velocity, 450 m/s; IMS wave height, 40 V; trap gas flow, 2 mL/min; IMS gas flow, 80 mL/min. Data were acquired and processed with MassLynx v4.1 and Driftscope version 2.8 software (Waters, Manchester, UK). Structural assignments were based on previously described IM-MS of BG505 SOSIP.664 glycans (Behrens et al., 2016) .
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