Protein Adsorption Loss─The Bottleneck of Single-Cell Proteomics

Abstract

Single-cell proteomics is a promising field to provide direct yet comprehensive molecular insights into cellular functions without averaging effects. Here, we address a grand technical challenge impeding the maturation of single-cell proteomicsprotein adsorption loss (PAL). Even though widely known, there is currently no quantitation on how profoundly and selectively PAL has affected single-cell proteomics. Therefore, the mitigations to this challenge have been generic, and their efficacy was only evaluated by the size of the resolved proteome with no specificity on individual proteins. We use the existing knowledge of PAL, protein expression, and the typical surface area used in single-cell proteomics to discuss the severity of protein loss. We also summarize the current solutions to this challenge and briefly review the available methods to characterize the physical and chemical properties of protein surface adsorption. By citing successful strategies in single-cell genomics for measurement errors in individual transcripts, we pinpoint the urgency to benchmark PAL at the proteome scale with individual protein resolution. Finally, orthogonal single-cell proteomic techniques that have the potential to cross validate PAL are proposed. We hope these efforts can promote the fruition of single-cell proteomics in the near future.

Proteomics to study cancer immunity and improve treatment

“…Single-cell proteomics is advancing rapidly [92,94,95], thanks to the continuous development of new MS instrumentation with increased sensitivity and specialized lossless sample preparation workflows. Nevertheless, protein adsorption loss during sample preparation still remains the main bottleneck for of single-cell proteomics [96], and optimized single-cell proteomics workflows therefore involves miniaturization to decrease interactions with hydrophobic surfaces, work in smallest possible volumes to keep samples as concentrated as possible and minimize buffer evaporation for sensitivity and reproducibility. Nowadays, with optimized single-cell proteomics workflows, it is possible to analyze ∼1000-3000 proteins from a single cell using label-free quantification [97] and isobaric labeling approaches [98], and up to 6,000 proteins from a few hundred cells [99].…”

Recent advances in microfluidics for single-cell functional proteomics

“…11 Yet, the feasibility and sensitivity of MS are significantly compromised at the single-cell level when using the traditional proteomics preparation workflows. 12 To address these limitations, enormous effort has thus been devoted to the miniaturization of sample preparations, optimization of analytical workflows, and integration of instruments, aiming to achieve a more streamlined SCP protocol with minimal sample-loss. Microfluidics, in particular, has been well-suited and employed for such endeavors.…”

“…11 Yet, the feasibility and sensitivity of MS are significantly compromised at the single-cell level when using the traditional proteomics preparation workflows. 12…”

Evaluation of the Binding Preference of Microtubes for Nanoproteomics Sample Preparation

“…One of the major challenges of this sample preparation process is the sample loss caused by protein/peptide absorption at the solid surface of the plastic container. 3 With most existing proteomics workflows, the use of polyethylene and polypropylene containers, mainly centrifuge or PCR microtubes, is largely unavoidable. However, it is well-known that these materials absorb proteins and peptides through various mechanisms, both reversibly and irreversibly.…”

“…Because of challenges in sample preparation, single-cell and nanoscale proteomics remain a daunting task. One of the major challenges of this sample preparation process is the sample loss caused by protein/peptide absorption at the solid surface of the plastic container . With most existing proteomics workflows, the use of polyethylene and polypropylene containers, mainly centrifuge or PCR microtubes, is largely unavoidable.…”