Collision energies: Optimization strategies for bottom‐up proteomics

Révész, Ágnes; Hevér, Helga; Steckel, Arnold; Schlosser, Gitta; Szabó, Dániel; Vékey, Károly; Drahos, László

doi:10.1002/mas.21763

Cited by 22 publications

(30 citation statements)

References 184 publications

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“…This approach is expected to provide several benefits, as some of us discussed in a recent review for other related analytes. 27 We further leveraged the resulting glycopeptidelevel information to design optimized measurement strategies, confirming the importance of both fine-tuning itself and using a diverse set of N-glycopeptide species for this purpose. Finally, our study is the first to provide reference data based on wellchosen standard materials for the transfer of the optimized method between mass spectrometers, which can guide scientists in choosing optimal settings on their experimental platforms in a laboratory or in a pharmaceutical industrial setting.…”

Section: ■ Introductionmentioning

confidence: 70%

“…In addition, though both studies used a QTof mass spectrometer from Bruker, instrumental differences may also contribute, as we have seen in the past for closely related Orbitrap instruments. 27 Different internal energy distribution on the two instruments, caused by differences of the ESI source (e.g., cone voltage and gas pressures) or voltages applied during ion transfer might be relevant candidates. 43−45 We note here that the different collision energy selection methods actually lead to perceivable differences at the level of individual peptides.…”

Section: Comparison To Literature Resultsmentioning

confidence: 99%

“… 12 As a result, various, somewhat different optimal methods were reported. 27 The obtained values are specific to the mass spectrometer used. In the case of Thermo Orbitrap instruments, an m / z -dependent normalized value is used (normalized collision energy, NCE), which is supposed to help in transferability of the settings.…”

Section: Introductionmentioning

confidence: 99%

“…However, it was found that different members of the Thermo Orbitrap equipment series still differ even in NCE terms. 27 The direct transfer and comparison of reported CE settings are even more difficult with QTof instruments, where “unnormalized” CE is applied explicitly.…”

Section: Introductionmentioning

confidence: 99%

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Diversity Matters: Optimal Collision Energies for Tandem Mass Spectrometric Analysis of a Large Set of N-Glycopeptides

et al. 2022

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Identification and characterization of N-glycopeptides from complex samples are usually based on tandem mass spectrometric measurements. Experimental settings, especially the collision energy selection method, fundamentally influence the obtained fragmentation pattern and hence the confidence of the database search results (“score”). Using standards of naturally occurring glycoproteins, we mapped the Byonic and pGlyco search engine scores of almost 200 individual N-glycopeptides as a function of collision energy settings on a quadrupole time of flight instrument. The resulting unprecedented amount of peptide-level information on such a large and diverse set of N-glycopeptides revealed that the peptide sequence heavily influences the energy for the highest score on top of an expected general linear trend with m/z. Search engine dependence may also be noteworthy. Based on the trends, we designed an experimental method and tested it on HeLa, blood plasma, and monoclonal antibody samples. As compared to the literature, these notably lower collision energies in our workflow led to 10–50% more identified N-glycopeptides, with higher scores. We recommend a simple approach based on a small set of reference N-glycopeptides easily accessible from glycoprotein standards to ease the precise determination of optimal methods on other instruments. Data sets can be accessed via the MassIVE repository (MSV000089657 and MSV000090218).

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Section: ■ Introductionmentioning

confidence: 70%

Section: Comparison To Literature Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Diversity Matters: Optimal Collision Energies for Tandem Mass Spectrometric Analysis of a Large Set of N-Glycopeptides

et al. 2022

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“…This suggested that larger proteoforms may need a higher energy for efficient backbone fragmentation. 45 A 22 kDa intact proteoform of Stathmin (STMN1, Uniprot#P16949) was shown as an example in Figure 5. The observed monoisotopic mass was 22358.55 Da with a mass measurement accuracy of 5.96 ppm compared with the theoretical mass of 22358.42 Da.…”

Section: Evaluation Of the Effect Of Normalized Hcd Fragmentation Ene...mentioning

confidence: 99%

Optimization of Higher-Energy Collisional Dissociation Fragmentation Energy for Intact Protein-Level Tandem Mass Tag Labeling

et al. 2023

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Isobaric chemical tag labeling (e.g., TMT) is a commonly used approach in quantitative proteomics, and quantification is enabled through detection of low-mass reporter ions generated after MS2 fragmentation. Recently, we have introduced and optimized an intact protein-level TMT labeling platform that demonstrated >90% labeling efficiency in complex samples with top-down proteomics. Higher-energy collisional dissociation (HCD) is commonly utilized for isobaric tag-labeled peptide fragmentation because it produces accurate reporter ion intensities and avoids loss of low mass ions. HCD energies have been optimized for isobaric tag labeled-peptides but have not been systematically evaluated for isobaric tag-labeled intact proteins. In this study, we report a systematic evaluation of normalized HCD fragmentation energies (NCEs) on TMT-labeled HeLa cell lysate using top-down proteomics. Our results suggested that reporter ions often result in higher ion intensities at higher NCEs. Optimal fragmentation of intact proteins for identification, however, required relatively lower NCE. We further demonstrated that a stepped NCE scheme with energies from 30% to 50% resulted in optimal quantification and identification of TMT-labeled HeLa proteins. These parameters resulted in an average reporter ion intensity of ∼4E4 and average proteoform spectrum matches (PrSMs) of >1000 per RPLC-MS/MS run with a 1% false discovery rate (FDR) cutoff.

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The use of sequential window acquisition of all theoretical fragment ion spectra (SWATH), a data‐independent acquisition high‐resolution mass spectrometry approach, in forensic toxicological regimes: A review

Sarkisian,

Rodda

2024

Drug Testing and Analysis

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Sequential window acquisition of all theoretical fragment ion spectra (SWATH) is a type of high‐resolution mass spectrometry that uses data‐independent acquisition. Compared with more targeted acquisition schemes, the power behind this data‐independent acquisition technique comes from its ability to mitigate interferences via the use of SWATH acquisition windows (Q1 quadrupole isolation windows) while still obtaining all accurate mass information. However, consistent with high‐resolution mass spectrometry techniques, its routine and high throughput implementation in forensic toxicology is limited due to the complex processing power required to effectively manage the large amount of acquired data. It is therefore pivotal to create an efficient and validated identification criterion that confidently reports suspected positive detections as a confirmational technique for final reporting. This review examines all publications that implemented SWATH in a forensic toxicological framework with suggestive best practices and commonly used criteria. Seventeen publications were reviewed for extraction, liquid chromatography and mass spectrometry parameters, and more specifically for all SWATH applicable characteristics including spray voltages, collision energies and spreads, mass error, isotopic ratio difference, retention time error, and library score thresholds. Notwithstanding the challenges SWATH implementation faces for a laboratory, the technique demonstrates its potential to be utilized in routine forensic toxicology testing regimes and aids in the detection of both common and emerging novel drugs simultaneously.

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Collision energies: Optimization strategies for bottom‐up proteomics

Cited by 22 publications

References 184 publications

Diversity Matters: Optimal Collision Energies for Tandem Mass Spectrometric Analysis of a Large Set of N-Glycopeptides

Diversity Matters: Optimal Collision Energies for Tandem Mass Spectrometric Analysis of a Large Set of N-Glycopeptides

Optimization of Higher-Energy Collisional Dissociation Fragmentation Energy for Intact Protein-Level Tandem Mass Tag Labeling

The use of sequential window acquisition of all theoretical fragment ion spectra (SWATH), a data‐independent acquisition high‐resolution mass spectrometry approach, in forensic toxicological regimes: A review

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