Evaluation of the Binding Preference of Microtubes for Nanoproteomics Sample Preparation

Abstract

Nonspecific binding between the protein and the container is an often-neglected cause of sample loss in large-scale proteomics sample preparation. In nanoproteomics, due to the small sample size, this absorption loss is no longer negligible, and researchers often adopt low binding plasticware to minimize the sample loss. However, there has been little discussion in the scientific literature on the differences in microtube performance on reducing protein/peptide binding. Therefore, the exact impact of sample loss during the sample preparation is not well understood. Here, we investigated the protein/peptide loss during the nanoproteomics experiment process. Our results showed that there are significant differences in nonspecific binding among the tested microtubes, with a protein recovery rate ranging from less than 10% to over 90% for different microtubes. Interestingly, we found that the storage temperature could also be one of the key factors that contribute to protein recovery from the plastic container. In addition, we investigated the binding preferences of different microtubes by the physical characteristics of the identified proteins and peptides, such as isoelectric point, hydrophobicity, length, and charge. Our findings help to better understand protein/peptide loss in proteomics sample preparation and provide further guidance for researchers in choosing proper containers for their precious sample.

Rapid, One-Step Sample Processing for Label-Free Single-Cell Proteomics

“…As these custom substrates are not yet widely available, we evaluated alternative nanowell substrates that were fabricated in polypropylene by injection molding. Polypropylene has been shown to minimize protein adsorption, 45 and injection molding can enable low-cost mass production for broader accessibility. We compared peptide yields from single HeLa cells prepared on either standard glass or hydrophobic injection-molded polypropylene nanowell chips using the one-step method (Figure 4a).…”

Silyl Ether Enables High Coverage Chemoproteomic Interaction Site Mapping

“…Using repeat injections of the same sample, we varied the MS2 resolving power from 15K to 60K, which revealed a modest increase in coverage for acquisition at 30K resolving power (Figure S3A). Motivated by recent reports of container dependent differences in coverage 57 , we compared the coverage obtained for samples prepared using microtubes from two different vendors and found that a 1.8-fold increase in peptide coverage could be achieved through container selection (Figure S3B). Building upon these coverage improvements, we then compared the coverage of the methyl 2a and ethyl 2b probes for datasets (Figure 2D).…”

Cheap, Robust and Versatile Two-Dimensional Chromatography system for Proteomics of Nanogram Scale Samples

“…19,20 Also, others have developed nano/microscale automated frac onator systems 18,21 , in an a empt to minimise losses during the transfer step of the frac ons to second dimension separa on which can also include the use of low-binding containers, low surface area glass nanowell chips and MS-compa ble addi ves in downstream sample handling steps. 19,21,22 A major challenge of frac ona on system is the need for bespoke and/or costly frac ona on equipment. Furthermore, robustness of nanoLC is substan ally worse than high flow systems and require constant monitoring for carryover and separa on performance.…”

“…19, 20 Also, others have developed nano/microscale automated fractionator systems 18, 21 , in an attempt to minimise losses during the transfer step of the fractions to second dimension separation which can also include the use of low-binding containers, low surface area glass nanowell chips and MS-compatible additives in downstream sample handling steps. 19, 21, 22…”