scholarly journals Benefits of Collisional Cross Section Assisted Precursor Selection (caps-PASEF) for Cross-linking Mass Spectrometry

2020 ◽  
Author(s):  
Barbara Steigenberger ◽  
Henk W.P. van den Toorn ◽  
Emiel Bijl ◽  
Jean-François Greisch ◽  
Oliver Räther ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Gas Phase ◽  
Cross Sections ◽  
Protein Interactions ◽  
Ion Mobility ◽  
Chemical Properties ◽  
Cross Linking ◽  
Mass Analyzer ◽  
Quadrupole Collision Cell ◽  
Collisional Cross Section

AbstractIon mobility separates molecules in the gas-phase on their physico-chemical properties, providing information about their size as collisional cross-sections. The timsTOF Pro combines trapped ion mobility with a quadrupole, collision cell and a time-of-flight mass analyzer, to probe ions at high speeds with on-the-fly fragmentation. Here, we show that on this platform ion mobility is beneficial for cross-linking mass spectrometry (XL-MS). Cross-linking reagents covalently link amino acids in close proximity, resulting in peptide pairs after proteolytic digestion. These cross-linked peptides are typically present at low abundance in the background of normal peptides, which can partially be resolved by using enrichable cross-linking reagents. Even with a very efficient enrichable cross-linking reagent, like PhoX, the analysis of cross-linked peptides is still hampered by the co-enrichment of peptides connected to a partially hydrolyzed reagent – termed mono-linked peptides. For experiments aiming to uncover protein-protein interactions these are unwanted byproducts. Here, we demonstrate that gas-phase separation by ion mobility enables the separation of mono-linked peptides from cross-linked peptide pairs. A clear partition between these two classes is observed at a CCS of 500 Å2 and a monoisotopic mass of 2 kDa, which can be used for targeted precursor selection. A total of 50 - 70% of the mono-linked peptides are prevented from sequencing, allowing the analysis to focus on sequencing the relevant cross-linked peptide pairs. In applications to both simple proteins and protein mixtures and a complete highly complex lysate this approach provides a substantial increase in detected cross-linked peptides.

scholarly journals Benefits of Collisional Cross Section Assisted Precursor Selection (caps-PASEF) for Cross-linking Mass Spectrometry

2020 ◽  
Vol 19 (10) ◽  
pp. 1677-1687 ◽  
Author(s):  
Barbara Steigenberger ◽  
Henk W. P. van den Toorn ◽  
Emiel Bijl ◽  
Jean-François Greisch ◽  
Oliver Räther ◽  
...  
Keyword(s):  
Gas Phase ◽  
Cross Sections ◽  
Protein Interactions ◽  
Ion Mobility ◽  
Chemical Properties ◽  
Cross Linking ◽  
Mass Analyzer ◽  
Protein Protein Interactions ◽  
Quadrupole Collision Cell ◽  
Collisional Cross Section

Ion mobility separates molecules in the gas-phase based on their physico-chemical properties, providing information about their size as collisional cross-sections. The timsTOF Pro combines trapped ion mobility with a quadrupole, collision cell and a TOF mass analyzer, to probe ions at high speeds with on-the-fly fragmentation. Here, we show that on this platform ion mobility is beneficial for cross-linking MS (XL-MS). Cross-linking reagents covalently link amino acids in proximity, resulting in peptide pairs after proteolytic digestion. These cross-linked peptides are typically present at low abundance in the background of normal peptides, which can partially be resolved by using enrichable cross-linking reagents. Even with a very efficient enrichable cross-linking reagent, like PhoX, the analysis of cross-linked peptides is still hampered by the co-enrichment of peptides connected to a partially hydrolyzed reagent – termed mono-linked peptides. For experiments aiming to uncover protein-protein interactions these are unwanted byproducts. Here, we demonstrate that gas-phase separation by ion mobility enables the separation of mono-linked peptides from cross-linked peptide pairs. A clear partition between these two classes is observed at a CCS of 500 Å2 and a monoisotopic mass of 2 kDa, which can be used for targeted precursor selection. A total of 50-70% of the mono-linked peptides are prevented from sequencing, allowing the analysis to focus on sequencing the relevant cross-linked peptide pairs. In applications to both simple proteins and protein mixtures and a complete highly complex lysate this approach provides a substantial increase in detected cross-linked peptides.

scholarly journals Cross-linking/Mass Spectrometry Combined with Ion Mobility on a timsTOF Pro Instrument for Structural Proteomics

2021 ◽  
Author(s):  
Christian H Ihling ◽  
Lolita Piersimoni ◽  
Marc Kipping ◽  
Andrea Sinz
Keyword(s):  
Mass Spectrometry ◽  
Cross Sections ◽  
Protein Interaction ◽  
Protein Interactions ◽  
Ion Mobility ◽  
Hek293t Cell ◽  
Structural Proteomics ◽  
Cross Linking ◽  
E Coli ◽  
Interaction Studies

The combination of cross-linking/mass spectrometry (XL-MS) and ion mobility is still underexplored for conducting protein conformational and protein-protein interaction studies. We present a method for analyzing cross-linking mixtures on a timsTOF Pro mass spectrometer that allows separating ions based on their gas phase mobilities. Cross-linking was performed with three urea-based MS-cleavable cross-linkers that deliver distinct fragmentation patterns for cross-linked species upon collisional activation. The discrimination of cross-linked species from non-cross-linked peptides was readily performed based on their collisional cross sections. We demonstrate the general feasibility of our combined XL-MS/ion mobility approach for three protein systems of increasing complexity: (i) Bovine serum albumin, (ii) E. coli ribosome, and (iii) HEK293T cell nuclear lysates. We identified a total of 508 unique cross-linking sites for BSA, 868 for the E. coli ribosome, and 1,623 unique cross-links for nuclear lysates, corresponding to 1,088 intra- and 535 interprotein interactions and yielding 564 distinct protein-protein interactions. Our results underline the strength of combining XL-MS with ion mobility not only for deriving 3D-structures of single proteins, but also for performing system-wide protein interaction studies.

scholarly journals Collision cross sections of high-mannose N-glycans in commonly observed adduct states – identification of gas-phase conformers unique to [M − H]− ions

The Analyst ◽  
2015 ◽  
Vol 140 (20) ◽  
pp. 6799-6803 ◽  
Author(s):  
W. B. Struwe ◽  
J. L. Benesch ◽  
D. J. Harvey ◽  
K. Pagel
Keyword(s):  
Mass Spectrometry ◽  
Gas Phase ◽  
Cross Sections ◽  
Ion Mobility ◽  
Drift Tube ◽  
Ion Mobility Mass Spectrometry ◽  
Collision Cross Sections ◽  
High Mannose

We report collision cross sections (CCS) of high-mannose N-glycans as [M + Na]+, [M + K]+, [M + H]+, [M + Cl]−, [M + H2PO4]− and [M − H]− ions, measured by drift tube (DT) ion mobility-mass spectrometry (IM-MS) in helium and nitrogen gases.

scholarly journals State-of-the-Art Native Mass Spectrometry and Ion Mobility Methods to Monitor Homogeneous Site-Specific Antibody-Drug Conjugates Synthesis

2021 ◽  
Vol 14 (6) ◽  
pp. 498
Author(s):  
Evolène Deslignière ◽  
Anthony Ehkirch ◽  
Bastiaan L. Duivelshof ◽  
Hanna Toftevall ◽  
Jonathan Sjögren ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Gas Phase ◽  
Ion Mobility ◽  
State Of The Art ◽  
Native Mass Spectrometry ◽  
Site Specific ◽  
Drug Conjugates ◽  
Conjugation Process ◽  
Antibody Drug

Antibody-drug conjugates (ADCs) are biotherapeutics consisting of a tumor-targeting monoclonal antibody (mAb) linked covalently to a cytotoxic drug. Early generation ADCs were predominantly obtained through non-selective conjugation methods based on lysine and cysteine residues, resulting in heterogeneous populations with varying drug-to-antibody ratios (DAR). Site-specific conjugation is one of the current challenges in ADC development, allowing for controlled conjugation and production of homogeneous ADCs. We report here the characterization of a site-specific DAR2 ADC generated with the GlyCLICK three-step process, which involves glycan-based enzymatic remodeling and click chemistry, using state-of-the-art native mass spectrometry (nMS) methods. The conjugation process was monitored with size exclusion chromatography coupled to nMS (SEC-nMS), which offered a straightforward identification and quantification of all reaction products, providing a direct snapshot of the ADC homogeneity. Benefits of SEC-nMS were further demonstrated for forced degradation studies, for which fragments generated upon thermal stress were clearly identified, with no deconjugation of the drug linker observed for the T-GlyGLICK-DM1 ADC. Lastly, innovative ion mobility-based collision-induced unfolding (CIU) approaches were used to assess the gas-phase behavior of compounds along the conjugation process, highlighting an increased resistance of the mAb against gas-phase unfolding upon drug conjugation. Altogether, these state-of-the-art nMS methods represent innovative approaches to investigate drug loading and distribution of last generation ADCs, their evolution during the bioconjugation process and their impact on gas-phase stabilities. We envision nMS and CIU methods to improve the conformational characterization of next generation-empowered mAb-derived products such as engineered nanobodies, bispecific ADCs or immunocytokines.

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