Enhanced Recovery of Low Concentration Protein and Peptide Solutions On Ultra-Low Binding Microplates

Weikart, Christopher M.; Breeland, Adam P.; Taha, Ahmad H.; Maurer, Brian R.

doi:10.4155/fsoa-2018-0099

Cited by 9 publications

(8 citation statements)

References 9 publications

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“…For example, reducing sample amounts without also paring preparation volumes can lead to an increased portion of the sample being lost to the surfaces of containers (e.g., well plates) due to nonspecific adsorption. [4][5][6][7] Similarly, the low resulting sample concentrations may experience decreased reaction rates for enzymatic and chemical reactions during processing. 8 Reducing sample-processing volumes can alleviate these issues and also save costs when precious reagents are used.…”

Section: Introductionmentioning

confidence: 99%

Adapting a Low-Cost and Open-Source Commercial Pipetting Robot for Nanoliter Liquid Handling

et al. 2021

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Section: Introductionmentioning

confidence: 99%

Adapting a Low-Cost and Open-Source Commercial Pipetting Robot for Nanoliter Liquid Handling

et al. 2021

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“…Furthermore, human serum proteins have a high propensity for binding polymers such as polypropylene and polystyrene [28,30], which form the basis of standard microcentrifuge tubes and microplates. Adsorption of proteins onto polymers is predominantly mediated via hydrophobic interactions [31]. Therefore, it is feasible that during the incubation of S. aureus with NHS, human serum proteins including C4BP can bind to the reaction vessels and remain following the washing stages.…”

Section: Discussionmentioning

confidence: 99%

Recruitment of C4b-binding protein is not a complement evasion strategy employed by Staphylococcus aureus

Li,

Bettoni,

Mohlin

et al. 2023

Microbiology

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Complement offers a first line of defence against infection through the opsonization of microbial pathogens, recruitment of professional phagocytes to the infection site and the coordination of inflammatory responses required for the resolution of infection. Staphylococcus aureus is a successful pathogen that has developed multiple mechanisms to thwart host immune responses. Understanding the precise strategies employed by S. aureus to bypass host immunity will be paramount for the development of vaccines and or immunotherapies designed to prevent or limit infection. To gain a better insight into the specific immune evasion mechanisms used by S. aureus we examined the pathogen’s interaction with the soluble complement inhibitor, C4b-binding protein (C4BP). Previous studies indicated that S. aureus recruits C4BP using a specific cell-wall-anchored surface protein and that bound C4BP limits complement deposition on the staphylococcal surface. Using flow-cytometric-based bacterial-protein binding assays we observed no interaction between S. aureus and C4BP. Moreover, we offer a precautionary warning that C4BP isolated from plasma can be co-purified with minute quantities of human IgG, which can distort binding analysis between S. aureus and human-derived proteins. Combined our data indicates that recruitment of C4BP is not a complement evasion strategy employed by S. aureus .

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“…Protein and peptide absorption on solid surfaces, e.g., glass and polymers, are well-known phenomena that have been studied extensively. [11][12][13][14] This is still often overlooked in larger-scale (>1 µg) proteomics experiments due to its uniform and negligible impact on protein identifications and quantitation. When it comes to single-cell proteomics with only picograms of protein samples dissolved in microliters of solvent, this absorption loss becomes no longer negligible and often dictate the outcome of the experiment.…”

Section: Introductionmentioning

confidence: 99%

Automated container-less cell processing method for single-cell proteomics

Matsumoto

Shao

Bogosavljevic

et al. 2022

Preprint

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Single-cell genomics and transcriptomics studies enabled us to characterize cell heterogeneity in various tissues, which helped us to better understand the biological system and disease progression. Single-cell proteomics, which directly measures the protein expression level, has the potential to further enhance our knowledge by providing not only a more direct measurement but also crucial information cannot be captured by genomics or transcriptomics study, such as protein activation states and post-translation modification events. One of the main challenges of single-cell proteomics is the large sample loss during sample preparation, which is largely unavoidable using standard proteomics protocols. Protein and peptide loss to the container surface is a well-known phenomenon but often overlooked in larger-scale (>1 microgram) proteomics experiments. When it comes to single-cell proteomics with only picograms of protein samples, this loss becomes non-negligible and often dictates the outcomes of the experiment. More importantly, sample processing through multiple pipette tips and containers often introduces random errors, which undermine the ability to detect true heterogenous cellular events. To solve these problems and further improve the throughput and reproducibility of the single-cell proteomics experiments, we developed an automated container-less cell processing platform, utilizing acoustic levitation to process cell samples in the air. Our platform automatically performs cell lysis, protein reduction, alkylation, digestion, and peptide labeling in the air, without any sample transfer step or container. The digested and labeled peptides are then directly injected into the capillary LC-MS/MS system for analysis, eliminating manual steps and conserving most of the sample materials for proteomics analysis. Our initial test shows at least 30% improvement in peptide signals over conventional methods. This process can be performed in parallel to further improve sample processing throughput.

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Enhanced Recovery of Low Concentration Protein and Peptide Solutions On Ultra-Low Binding Microplates

Cited by 9 publications

References 9 publications

Adapting a Low-Cost and Open-Source Commercial Pipetting Robot for Nanoliter Liquid Handling

Adapting a Low-Cost and Open-Source Commercial Pipetting Robot for Nanoliter Liquid Handling

Recruitment of C4b-binding protein is not a complement evasion strategy employed by Staphylococcus aureus

Automated container-less cell processing method for single-cell proteomics

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