Initial recommendations for performing, benchmarking and reporting single-cell proteomics experiments

Gatto, Laurent; Aebersold, Ruedi; Cox, Jürgen; Demichev, Vadim; Derks, Jason; Emmott, Edward; Franks, Alexander; Ivanov, Alexander R.; Kelly, Ryan; Khoury, Luke; Leduc, Andrew; MacCoss, Michael J.; Nemes, Péter; Perlman, David H.; Petelski, Aleksandra A.; Rose, Christopher M.; Schoof, Erwin M.; Eyk, Jennifer E. Van; Vanderaa, Christophe; Yates, John R.; Slavov, Nikolai

doi:10.1038/s41592-023-01785-3

Cited by 79 publications

(104 citation statements)

References 93 publications

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“…Such pooling can enhance peptide sequence identification analogously to the pooling that happens with isobaric carriers, , but it also may limit the specificity of sample-specific sequence identification. Thus, rigorous models of amino acid sequence identification should ensure robust FDR estimations and benchmark them with mixed species experiments as described in the community white paper on single-cell proteomics …”

Section: Opportunities For Advancing Plexdiamentioning

confidence: 99%

“…This capacity limitation is likely to be more pronounced for DIA analysis using isotopologous carriers because of the wider isolation windows used by DIA. Thus, the development of isotopologous carriers can be informed by established principles for the use of carriers, , and isotopologous carriers compatible with accurate and precise quantification are likely to be smaller than isobaric carriers. Thus, the optimal carrier size and data acquisition methods should be rigorously benchmarked using mixed species experiments as with the development of plexDIA …”

Section: Opportunities For Advancing Plexdiamentioning

confidence: 99%

“…Thus, rigorous models of amino acid sequence identification should ensure robust FDR estimations and benchmark them with mixed species experiments as described in the community white paper on single-cell proteomics. 4 Increasing the Number of Multiplexed Samples. The multiplicative scaling of throughput by plexDIA has been demonstrated with a 3-plexDIA, 16 and we expect this framework to extend to suitably engineered higher plex mass tags.…”

Section: Developing Mass-tags Optimized For Plexdiamentioning

confidence: 99%

“…54 Thus, such extensions of plexDIA hold the potential of extending the toolset of singlecell analysis to functional assays quantifying protein shapes and activities. 4,50,58 ■ METHODS…”

Section: Functional Proteomic Assays With Plexdiamentioning

confidence: 99%

“…Tandem mass-spectrometry (MS) has long been established as the most specific, comprehensive, and versatile method for protein analysis. − However, the sensitivity and throughput of MS have traditionally limited the biomedical applications of MS proteomics. These limitations are increasingly mitigated by new approaches that increase the sensitivity − and throughput , of MS-based proteomics. Many of these advances take advantage of data independent acquisition (DIA), which was introduced decades ago and has developed into powerful methodologies. − …”

Section: Introductionmentioning

confidence: 99%

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

Derks

Slavov

2023

J. Proteome Res.

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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.

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Section: Opportunities For Advancing Plexdiamentioning

confidence: 99%

Section: Opportunities For Advancing Plexdiamentioning

confidence: 99%

Section: Developing Mass-tags Optimized For Plexdiamentioning

confidence: 99%

“…54 Thus, such extensions of plexDIA hold the potential of extending the toolset of singlecell analysis to functional assays quantifying protein shapes and activities. 4,50,58 ■ METHODS…”

Section: Functional Proteomic Assays With Plexdiamentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

Derks

Slavov

2023

J. Proteome Res.

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Artificial Breakthrough of Cell Membrane Barrier for Transmembrane Substance Exchange: A Review of Recent Progress

Wang,

Zhu,

et al. 2023

Adv Funct Materials

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Cell membrane composed of lipid bilayer is a selectively permeable membrane that only allows specific molecules to pass through. While such selectivity is essential for the survival and function of cells, there exist instances when it is necessary to overcome the cell membrane barrier. Study on the artificial cell membrane barrier breakthrough strategies is of great significance for the development of drug delivery systems and the understanding of cellular behaviors. Herein, the advancements in the development of strategies for opening cell membrane barriers over the past decade are summarized. The main transmembrane mechanisms are elucidated and then divided into three categories, i.e., cell perforation via microinjection/external physical fields, cell endocytosis‐assisted construction of artificial transmembrane channels, and untethered micro/nanomachines. Next, the potential applications after opening the cell membrane are discussed, which mainly focus on the transmembrane cargo delivery into the cell and endogenous substance extraction out from the cell. Finally, this review outlines the current challenges that impede the realization of practical applications and presents an outlook of future opportunities to promote further development. Through overcoming the challenges, it is anticipated that artificial cell membrane breakthrough strategies will provide a revolutionized tool in the near future to advance the field of biomedicine and biotechnology.

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Boronic Acid‐Rich Lanthanide Metal‐Organic Frameworks Enable Deep Proteomics with Ultratrace Biological Samples

Zhang,

Ghalandari,

Chen

et al. 2024

Advanced Materials

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Label‐free proteomics is widely used to identify disease mechanism and potential therapeutic targets. However, deep proteomics with ultratrace clinical specimen remains a major technical challenge due to extensive contact loss during complex sample pretreatment. Here, a hybrid of four boronic acid‐rich lanthanide metal‐organic frameworks (MOFs) with high protein affinity is introduced to capture proteins in ultratrace samples jointly by nitrogen‐boronate complexation, cation‐π and ionic interactions. A MOFs Aided Sample Preparation (MASP) workflow that shrinks sample volume and integrates lysis, protein capture, protein digestion and peptide collection steps into a single PCR tube to minimize sample loss caused by non‐specific absorption, is proposed further. MASP is validated to quantify ≈1800 proteins in 10 HEK‐293T cells. MASP is applied to profile cerebrospinal fluid (CSF) proteome from cerebral stroke and brain damaged patients, and identified ≈3700 proteins in 1 µL CSF. MASP is further demonstrated to detect ≈9600 proteins in as few as 50 µg mouse brain tissues. MASP thus enables deep, scalable, and reproducible proteome on precious clinical samples with low abundant proteins.

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Initial recommendations for performing, benchmarking and reporting single-cell proteomics experiments

Cited by 79 publications

References 93 publications

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

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

Artificial Breakthrough of Cell Membrane Barrier for Transmembrane Substance Exchange: A Review of Recent Progress

Boronic Acid‐Rich Lanthanide Metal‐Organic Frameworks Enable Deep Proteomics with Ultratrace Biological Samples

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