Longitudinal Assessment of Peptide Recoveries from a Sample Solution in an Autosampler Vial for Proteomics

Abstract

Relatively few studies on time-related peptide losses have been reported and previous studies on the differential peptide recovery from a sample solution over a period had limitations. Thus, here, longitudinal peptide losses during sample solution storage in an autosampler vial over 145 h were assessed using nanoliquid chromatography and tandem mass spectrometry. In this study, hydrophilic peptide losses from a solution stored in a vial were observed regardless of materials of vials and the possibility of hydrophobic peptide losses brought by reasons other than hydrophobic peptide adsorption onto vials was found. Additionally, the preparation of a proteomics sample using 0.1% aqueous formic acid solution (0.1% FA) and a polypropylene vial, 0.1% FA and a glass vial, or the aqueous mixture of 5 M urea, 1 M thiourea, and 0.1% FA and a polymethylpentene vial proved to produce the most reliable longitudinal recovery of peptides from a solution stored in a vial.

Ultrasensitive Trace Sample Proteomics Unraveled the Protein Remodeling during Mesenchymal–Amoeboid Transition

“…Protein digestion is the most time-consuming part in the proteomic pipeline, which always takes overnight incubation . While the inner wall of vail was reported to adsorb the digested peptides, − and the over-digestion leads to transpeptidation; therefore, long time incubation may be detrimental for the recovery of peptides for trace samples. To accelerate the digestion process, surfactants such as RapiGest and DDM were reported to be functional in trace sample analysis. ,, With the aid of RapiGest, interestingly, during the digestion process of 2 h to overnight, an average of ∼5000 protein groups and ∼47 000 peptides were detected in 1000 HeLa cells, while the number of identifications after overnight digestion was slightly lower than 2 h digestion with no bias (Figures A and S2).…”

“…Multiple efforts have been made to improve protein and peptide recoveries for limited samples, from sample collection to liquid chromatography–mass spectrometry (LC–MS/MS) analysis . The main obstacle in minute sample analysis is the sample loss and contaminants during the multistep sample processing, as well as protein purification aiming to the removal of LC–MS-incompatible detergents. , Besides, nonspecific adsorption of the proteins and peptides to vial walls , during sample pretreatment and transfer, as well as specific adsorption by hydrophilic-coated tubes, also caused sample wastage.…”

Capillary Electrophoresis Coupled to Electrospray Ionization Tandem Mass Spectrometry for Ultra-Sensitive Proteomic Analysis of Limited Samples

“…Thus, the numbers of identifications in analyses following the first one dropped by approximately 63, 54, 28, and 56% for proteins, peptides groups, phosphopeptides, and acetylated peptides, respectively. These results suggest that protein is lost over time, likely due to adsorption to the plastic walls of the sample vial. , Alternative materials used for sample vials are needed to avoid such losses in the future. Vials made from glass or with lower surface area contact with the sample, for example, result in less non-specific adsorption to vessel walls. , Modified peptides were also monitored in these experiments, showing averages of 4 ± 3, 33 ± 6, and 41 ± 3 phosphopeptides and 103 ± 32, 391 ± 23, and 439 ± 12 N-terminal acetylated peptides from 1, 5, and 10 HeLa cells, respectively (Figure D).…”

“…These results suggest that protein is lost over time, likely due to adsorption to the plastic walls of the sample vial. 40,41 Alternative materials used for sample vials are needed to avoid such losses in the future. Vials made from glass or with lower surface area contact with the sample, for example, result in less non-specific adsorption to vessel walls.…”

Simple and Efficient Microsolid-Phase Extraction Tip-Based Sample Preparation Workflow to Enable Sensitive Proteomic Profiling of Limited Samples (200 to 10,000 Cells)

“…Sample processing for bottom-up proteomics is a multistep procedure, typically consisting of cell/tissue lysis, protein extraction and denaturation, disulfide bond reduction, cysteine alkylation, and proteolytic digestion steps. The main challenges preventing efficient recovery of peptides available at scarce amounts are nonspecific adsorption to exposed surfaces during these multiple sample processing steps and required sample transfers , and losses during cleanup steps that include the removal of incompatible salts and detergents . Several groups have reported on different strategies to improve recovery of limited biological samples, including using chemically passivated or low retention surfaces, ,, minimizing sample volume and contact surface area, ,,− and integrating contaminant removal devices so that all steps can be performed within a single vessel. ,− For samples >1000 cells, the most widely used protocols are in-StageTip (iST) sample processing, filter-aided sample preparation (FASP), sample processing within a kinked microreactor tip, and single-pot solid-phase-enhanced sample preparation (SP3) .…”