Estimating Total Quantitative Protein Content in Escherichia coli, Saccharomyces cerevisiae, and HeLa Cells

Dolgalev, Georgii; Safonov, Taras A.; Arzumanian, Viktoriia A.; Kiseleva, Olga I.; Poverennaya, Ekaterina V.

doi:10.3390/ijms24032081

Cited by 7 publications

(5 citation statements)

References 56 publications

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“…Interestingly, in shorter gradient times the impact of window size on %CV is less pronounced, particularly with the 11-minute gradient where there is little difference in the two window sizes, and in general shorter gradients have tighter CV distributions. For HeLa, a 45-minute gradient covers approximately 90% of the expressed proteome, 16 while an 8-minute gradient can deliver 78% as many hits in less than 1/5 of the time. In terms of copy numbers per cell, this level of coverage can observe proteins at ~100 copies, with the lowest observed protein at an estimated 13 copies per cell (supplementary figure 1).…”

Section: Resultsmentioning

confidence: 99%

An inflection point in high-throughput proteomics with Orbitrap Astral: analysis of biofluids, cells, and tissues

Hendricks,

Bhosale,

Keoseyan

et al. 2024

Preprint

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This technical note presents a comprehensive proteomics workflow for the new combination of Orbitrap and Astral mass analyzers across biofluids, cells, and tissues. Central to our workflow is the integration of Adaptive Focused Acoustics (AFA) technology for cells and tissue lysis, to ensure robust and reproducible sample preparation in a high-throughput manner. Furthermore, we automated the detergent-compatible single-pot, solid-phase-enhanced sample Preparation (SP3) method for protein digestion, a technique that streamlines the process by combining purification and digestion steps, thereby reducing sample loss and improving efficiency. The synergy of these advanced methodologies facilitates a robust and high-throughput approach for cells and tissue analysis, an important consideration in translational research. This work disseminates our platform workflow, analyzes the effectiveness, demonstrates reproducibility of the results, and highlights the potential of these technologies in biomarker discovery and disease pathology. For cells and tissues (heart, liver, lung, and intestine) proteomics analysis by data-independent acquisition mode, identifications exceeding 10,000 proteins can be achieved with a 24-minute active gradient. In 200ng injections of HeLa digest across multiple gradients, an average of more than 80% of proteins have a CV less than 20%, and a 45-minute run covers ~90% of the expressed proteome. In plasma samples including naive, depleted, perchloric acid precipitated, and Seer nanoparticle captured, all with a 24-minute gradient length, we identified 87, 108, 96 and 137 out of 216 FDA approved circulating protein biomarkers, respectively. This complete workflow allows for large swaths of the proteome to be identified and is compatible across diverse sample types.

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Section: Resultsmentioning

confidence: 99%

An inflection point in high-throughput proteomics with Orbitrap Astral: analysis of biofluids, cells, and tissues

Hendricks,

Bhosale,

Keoseyan

et al. 2024

Preprint

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show abstract

“…55 The system was able to load 40% of the available peptide material in each sample vial for each measurement, corresponding to the injection of 0.1−10 ng of the HeLa cell digest. If we assume each HeLa cell contains roughly 250 pg proteins, 56,57 the mass of available peptide material in each sample vial corresponds to roughly 1−100 cells. The mass of injected peptides equals that in only 0.4−40 cells.…”

Section: ■ Results and Discussionmentioning

confidence: 99%

Solid-Phase Microextraction-Aided Capillary Zone Electrophoresis-Mass Spectrometry: Toward Bottom-Up Proteomics of Single Human Cells

Colón Rosado,

Sun

2024

J. Am. Soc. Mass Spectrom.

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Capillary zone electrophoresis-mass spectrometry (CZE-MS) has been recognized as a valuable technique for the proteomics of mass-limited biological samples (i.e., single cells). However, its broad adoption for single cell proteomics (SCP) of human cells has been impeded by the low sample loading capacity of CZE, only allowing us to use less than 5% of the available peptide material for each measurement. Here we present a reversed-phase-based solid-phase microextraction (RP-SPME)-CZE-MS platform to solve the issue, paving the way for SCP of human cells using CZE-MS. The RP-SPME-CZE system was constructed in one fused silica capillary with zero dead volume for connection via in situ synthesis of a frit, followed by packing C8 beads into the capillary to form a roughly 2 mm long SPME section. Peptides captured by SPME were eluted with a buffer containing 30% (v/v) acetonitrile and 50 mM ammonium acetate (pH 6.5), followed by dynamic pH junction-based CZE-MS. The SPME-CZE-MS enabled the injection of nearly 40% of the available peptide sample for each measurement. The system identified 257 ± 24 proteins and 523 ± 69 peptides (N = 2) using a Q-Exactive HF mass spectrometer when only 0.25 ng of a commercial HeLa cell digest was available in the sample vial and 0.1 ng of the sample was injected. The amount of available peptide is equivalent to the protein mass of one HeLa cell. The data indicate that SPME-CZE-MS is ready for SCP of human cells.

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“…Such a feature would be unexpected for a strategy I podovirus that depends on an OM protein for infection. The NGR receptor is present in small amounts on the cell surface because only about 45 copies of the NfrA protein (equal to five to six complexes) are present per cell [84]. At the same time, this polysaccharide is likely to be conserved among many E. coli strains, similar to enterobacterial common antigen (ECA).…”

Section: Podoviruses: Cut or Pull?mentioning

confidence: 99%

Bacterial Virus Forcing of Bacterial O-Antigen Shields: Lessons from Coliphages

Letarov

2023

IJMS

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In most Gram-negative bacteria, outer membrane (OM) lipopolysaccharide (LPS) molecules carry long polysaccharide chains known as the O antigens or O polysaccharides (OPS). The OPS structure varies highly from strain to strain, with more than 188 O serotypes described in E. coli. Although many bacteriophages recognize OPS as their primary receptors, these molecules can also screen OM proteins and other OM surface receptors from direct interaction with phage receptor-binding proteins (RBP). In this review, I analyze the body of evidence indicating that most of the E. coli OPS types robustly shield cells completely, preventing phage access to the OM surface. This shield not only blocks virulent phages but also restricts the acquisition of prophages. The available data suggest that OPS-mediated OM shielding is not merely one of many mechanisms of bacterial resistance to phages. Rather, it is an omnipresent factor significantly affecting the ecology, phage–host co-evolution and other related processes in E. coli and probably in many other species of Gram-negative bacteria. The phages, in turn, evolved multiple mechanisms to break through the OPS layer. These mechanisms rely on the phage RBPs recognizing the OPS or on using alternative receptors exposed above the OPS layer. The data allow one to forward the interpretation that, regardless of the type of receptors used, primary receptor recognition is always followed by the generation of a mechanical force driving the phage tail through the OPS layer. This force may be created by molecular motors of enzymatically active tail spikes or by virion structural re-arrangements at the moment of infection.

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Estimating Total Quantitative Protein Content in Escherichia coli, Saccharomyces cerevisiae, and HeLa Cells

Cited by 7 publications

References 56 publications

An inflection point in high-throughput proteomics with Orbitrap Astral: analysis of biofluids, cells, and tissues

An inflection point in high-throughput proteomics with Orbitrap Astral: analysis of biofluids, cells, and tissues

Solid-Phase Microextraction-Aided Capillary Zone Electrophoresis-Mass Spectrometry: Toward Bottom-Up Proteomics of Single Human Cells

Bacterial Virus Forcing of Bacterial O-Antigen Shields: Lessons from Coliphages

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