Data-Driven Optimization of DIA Mass Spectrometry by DO-MS

Wallmann, Georg; Leduc, Andrew; Slavov, Nikolai

doi:10.1101/2023.02.02.526809

Cited by 3 publications

(5 citation statements)

References 56 publications

(127 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The optimization of MS data acquisition parameters for single-cell analysis can be performed with standards and has been detailed in ref. 39,56,57 . After this optimization, the success of the sample preparation can be assessed with the quality control (QC) reports generated by the QuantQC package in R. The full QuantQC report can be found in supplemental file 1, and several plots are highlighted in Fig.…”

Section: Anticipated Resultsmentioning

confidence: 99%

“…Optimization of LC/MS setup for single-cell protein analysis has been discussed extensively elsewhere 39,56,57 . Describing our setup briefly, TMT multiplexed data was run on an Exploris 480 mass spectrometer with the Neo Vanquish LC system and 25 cm X 75 μm ID IonOpticks Aurora separation column.…”

Section: Lc/ms Setupmentioning

confidence: 99%

“…The analytical column used was a 25 cm X 75 μm ID IonOpticks Aurora column with a captive spray fitting. The plexDIA data acquisition was performed using a method with 100ms fill times and 8 PASEF frames per duty cycle, with an additional MS1 frame after every 2 MS2 frames to improve the MS1 duty cycle 17,26,57 . The LC gradient on the NanoElute2 ramped from 4 to 38% buffer B and peptides eluted for 20 minutes, with a 45 minute total run time.…”

Section: Lc/ms Setupmentioning

confidence: 99%

See 2 more Smart Citations

Massively parallel sample preparation for multiplexed single-cell proteomics using nPOP

Leduc,

Koury,

Cantlon

et al. 2023

Preprint

Self Cite

View full text Add to dashboard Cite

Single-cell proteomics by mass spectrometry (MS) allows quantifying proteins with high specificity and sensitivity. To increase its throughput, we developed nPOP, a method for parallel preparation of thousands of single cells in nanoliter volume droplets deposited on glass slides. Here, we describe its protocol with emphasis on its flexibility to prepare samples for different multiplexed MS methods. An implementation with plexDIA demonstrates accurate quantification of about 3,000 - 3,700 proteins per human cell. The protocol is implemented on the CellenONE instrument and uses readily available consumables, which should facilitate broad adoption. nPOP can be applied to all samples that can be processed to a single-cell suspension. It takes 1 or 2 days to prepare over 3,000 single cells. We provide metrics and software for quality control that can support the robust scaling of nPOP to higher plex reagents for achieving reliable high-throughput single-cell protein analysis.

show abstract

Section: Anticipated Resultsmentioning

confidence: 99%

Section: Lc/ms Setupmentioning

confidence: 99%

Section: Lc/ms Setupmentioning

confidence: 99%

See 1 more Smart Citation

Massively parallel sample preparation for multiplexed single-cell proteomics using nPOP

Leduc,

Koury,

Cantlon

et al. 2023

Preprint

Self Cite

View full text Add to dashboard Cite

show abstract

“…Furthermore, duty cycle parameters can be algorithmically optimized to distribute ions evenly across isolation windows and thus reduce overcrowding of ions in some windows and the associated potential for increased interference. Such optimization can be performed with multiple tools, including with DO-MS, 41,42 which provides support for plexDIA. Sequence propagation has long been fruitfully applied across different runs using a variety of software tools.…”

Section: Plexdia Algorithms For Enhanced Sequence Identificationmentioning

confidence: 99%

Strategies for Increasing the Depth and Throughput of Protein Analysis by plexDIA

Derks

Slavov

2023

J. Proteome Res.

Self Cite

View full text Add to dashboard Cite

Accurate protein quantification is key to identifying protein markers, regulatory relationships between proteins, and pathophysiological mechanisms. Realizing this potential requires sensitive and deep protein analysis of a large number of samples. Toward this goal, proteomics throughput can be increased by parallelizing the analysis of both precursors and samples using multiplexed data independent acquisition (DIA) implemented by the plexDIA framework: https://plexDIA.slavovlab.net. Here we demonstrate the improved precisions of retention time estimates within plexDIA and how this enables more accurate protein quantification. plexDIA has demonstrated multiplicative gains in throughput, and these gains may be substantially amplified by improving the multiplexing reagents, data acquisition, and interpretation. We discuss future directions for advancing plexDIA, which include engineering optimized mass-tags for high-plexDIA, introducing isotopologous carriers, and developing algorithms that utilize the regular structures of plexDIA data to improve sensitivity, proteome coverage, and quantitative accuracy. These advances in plexDIA will increase the throughput of functional proteomic assays, including quantifying protein conformations, turnover dynamics, modifications states and activities. The sensitivity of these assays will extend to single-cell analysis, thus enabling functional single-cell protein analysis.

show abstract

Section: Opportunities For Advancing Plexdiamentioning

confidence: 99%

Strategies for increasing the depth and throughput of protein analysis by plexDIA

Derks

Slavov

2022

Preprint

View full text Add to dashboard Cite

Accurate protein quantification is key to identifying protein markers, regulatory relationships between proteins, and pathophysiological mechanisms. Realizing this potential requires sensitive and deep protein analysis of a large number of samples. Toward this goal, proteomics throughput can be increased by parallelizing the analysis of both precursors and samples using multiplexed data independent acquisition (DIA) implemented by the plexDIA framework. Here we demonstrate the improved precisions of RT estimates within plexDIA and how this enables more accurate protein quantification. plexDIA has demonstrated multiplicative gains in throughput, and these gains may be substantially amplified by improving the multiplexing reagents, data acquisition and interpretation. We discuss future directions for advancing plexDIA, which include engineering optimized mass-tags for high-plexDIA and developing algorithms that utilize the regular structures of plexDIA data to improve sensitivity, proteome coverage and quantitative accuracy. These advances in plexDIA will increase the throughput of functional proteomic assays, including quantifying protein conformations, turnover dynamics, modifications states and activities. The sensitivity of these assays will extend to single-cell analysis, thus enabling functional single-cell protein analysis.

show abstract

Data-Driven Optimization of DIA Mass Spectrometry by DO-MS

Cited by 3 publications

References 56 publications

Massively parallel sample preparation for multiplexed single-cell proteomics using nPOP

Massively parallel sample preparation for multiplexed single-cell proteomics using nPOP

Strategies for Increasing the Depth and Throughput of Protein Analysis by plexDIA

Strategies for increasing the depth and throughput of protein analysis by plexDIA

Contact Info

Product

Resources

About