Ion Activation Methods for Peptides and Proteins

Macias, Luis A.; Santos, Inês C.; Brodbelt, Jennifer S.

doi:10.1021/acs.analchem.9b04859

Cited by 87 publications

(121 citation statements)

References 502 publications

(1,243 reference statements)

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“…Tandem mass spectrometry has become a widely used analytical tool in the investigation of small molecules as well as in proteomics. The applied methods involve various fragmentation techniques differing in the mode of activation, for example, collision‐induced dissociation (CID) or electron transfer dissociation (ETD) 1–3 . In the case of the most frequently used CID, spectrum characteristics are mainly influenced by energetics (like low and high energy CID), by the timescale, and the number of collisions (like quadrupoles or ion traps) but also depend on instrumentation (like triple quadrupole [QQQ] or quadrupole time‐of‐flight [QTof]) and on experimental conditions.…”

Section: Introductionmentioning

confidence: 99%

Collision energies on QTof and Orbitrap instruments: How to make proteomics measurements comparable?

et al. 2020

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Quadrupole time‐of‐flight (QTof) collision‐induced dissociation (CID) and Orbitrap higher‐energy collisional dissociation (HCD) are the most commonly used fragmentation techniques in mass spectrometry‐based proteomics workflows. The information content of the MS/MS spectra is first and foremost determined by the applied collision energy. How can we set up the two instrument types to achieve maximum transferability? To answer this question, we compared MS/MS spectra obtained on a Bruker QTof CID and a Thermo Q‐Exactive Focus Orbitrap HCD instrument as a function of collision energy using the similarity index. Results show that with a few eV lower collision energy setting on HCD (Orbitrap‐specific CID) than on QTof CID, nearly identical MS/MS spectra can be obtained for leucine enkephalin pentapeptide standard, for selected +2 and +3 enolase tryptic peptides and for a large number of peptides in a HeLa protein digest. The Bruker QTof was able to produce colder ions, which may be significant to study inherently labile compounds. Further, we examined energy dependence of peptide identification confidence, as characterized by Mascot scores, on the HeLa peptides. In line with earlier QTof results, this dependence shows one or two maxima (unimodal or bimodal behavior) on Orbitrap. The fraction of bimodal peptides is lower on Orbitrap. Optimal energies as a function of m/z show a similar linear trend on both instruments, which suggests that with appropriate collision energy adjustment, matching conditions for proteomics can be achieved. Data have been deposited in the MassIVE repository (MSV000086434).

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Section: Introductionmentioning

confidence: 99%

Collision energies on QTof and Orbitrap instruments: How to make proteomics measurements comparable?

et al. 2020

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“…Most frequently, peptide ions are fragmented by collisions with inert gas molecules (collision-induced dissociation, CID). 11 The conventional data acquisition strategy is the data-dependent analysis (DDA) involving mass selection and MS/MS measurement of the most abundant precursors present in a preceding MS scan. The resulting fragment ion spectra of individual peptides are then searched against a sequence database to identify the peptide sequence, and proteins are inferred from the identified peptides.…”

Section: Introductionmentioning

confidence: 99%

Tailoring to Search Engines: Bottom-Up Proteomics with Collision Energies Optimized for Identification Confidence

et al. 2020

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Bottom-up proteomics relies on identification of peptides from tandem mass spectra, usually via matching against sequence databases. Confidence in a peptide−spectrum match can be characterized by a score value given by the database search engines, and it depends on the information content and the quality of the spectrum. The latter are influenced by experimental parameters, of which the collision energy is the most important one in the case of collision-induced dissociation. We examined how the identification score of the Byonic and Andromeda (MaxQuant) engines varies with collision energy for more than a thousand individual peptides from a HeLa tryptic digest on a QTof instrument. We thereby extended our earlier study on Mascot scores and corroborated its findings on the potential bimodal nature of this energy dependence. Optimal energies as a function of m/z show comparable linear trends for the three engines. On the basis of peptidelevel results, we designed methods with one or two liquid chromatography−tandem mass spectrometry (LC-MS/MS) runs and various collision energy settings and assessed their practical performance in peptide and protein identification from the HeLa standard sample. A 10−40% gain in various measures, such as the number of identified proteins or sequence coverage, was obtained over the factory default settings. Best performing methods differ for the three engines, suggesting that the experimental parameters should be fine-tuned to the choice of the engine. We also recommend a simple approach and provide reference data to ease the transfer of the optimized methods to other mass spectrometers relevant for proteomics. We demonstrate the utility of this approach on an Orbitrap instrument. Data sets can be accessed via the MassIVE repository (MSV000086379).

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“…Even the best-suited enrichment methods lack utility if the glycopeptides they enrich cannot be readily identified with available MS acquisition and informatics tools. Additionally, ETD and ECD methods often benefit from hybrid approaches that use supplemental activation either during or following the electron-driven dissociation event to promote more by guest on October 30, 2020 extensive fragmentation (482,483). These hybrid methods have been shown to improve both Nand O-glycopeptide characterization (322,(484)(485)(486)(487)(488)(489)(490), but they are effectively required for sitespecific analyses of O-glycopeptides (52,113,114,306,475).…”

Section: Related Developments In Glycoproteomicsmentioning

confidence: 99%

A Pragmatic Guide to Enrichment Strategies for Mass Spectrometry–Based Glycoproteomics

Riley

Bertozzi

Pitteri

2021

Molecular & Cellular Proteomics

165

158

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Glycosylation is a prevalent, yet heterogeneous modification with a broad range of implications in molecular biology. This heterogeneity precludes enrichment strategies that can be universally beneficial for all glycan classes. Thus, choice of enrichment strategy has profound implications on experimental outcomes. Here we review common enrichment strategies used in modern mass spectrometry (MS)-based glycoproteomic experiments, including lectins and other affinity chromatographies, hydrophilic interaction chromatography (HILIC) and its derivatives, porous graphitic carbon (PGC), reversible and irreversible chemical coupling strategies, and chemical biology tools that often leverage bioorthogonal handles. Interest in glycoproteomics continues to surge as MS instrumentation and software improve, so this review aims to help equip researchers with necessary information to choose appropriate enrichment strategies that best complement these efforts.

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Ion Activation Methods for Peptides and Proteins

Cited by 87 publications

References 502 publications

Collision energies on QTof and Orbitrap instruments: How to make proteomics measurements comparable?

Collision energies on QTof and Orbitrap instruments: How to make proteomics measurements comparable?

Tailoring to Search Engines: Bottom-Up Proteomics with Collision Energies Optimized for Identification Confidence

A Pragmatic Guide to Enrichment Strategies for Mass Spectrometry–Based Glycoproteomics

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