Optimization on Single-Pot, Solid-Phase-Enhanced Sample Preparation Method for Proteomics Workflow

Mass spectrometry-based proteomics relies on effective sample preparation to achieve accurate protein identification and quantification. Among current methods, Single-Pot, Solid-Phase-Enhanced Sample Preparation (SP3) is widely used because of its simple workflow, compatibility with different buffer systems, and suitability for low-input samples. However, key aspects of SP3, including reproducibility and sample loss during workflow, remain unclear. The primary objective of this work was to quantitatively assess peptide recovery after SP3 digestion under different experimental conditions. Controlled studies were performed using BSA as a model protein. Peptide concentrations were measured using the BCA peptide assay, with assay sensitivity evaluated through Limit of Detection (LOD) and Limit of Quantification (LOQ) values. Statistical analyses were applied to examine recovery and reproducibility across replicates using mean and standard deviation. In general, sample groups consistently showed higher recovery than their controls, confirming that SP3 can effectively process proteins, although some loss still occurred. Recovery varied between conditions, with some generating moderate yields and others much higher peptide recovery. Protein loss during the SP3 workflow was further examined, particularly during the binding and washing steps. NHS-activated Sepharose beads were tested as a possible strategy to capture unbound proteins from supernatant and washing fractions. However, comparisons between activated NHS beads and controls showed minimal differences, suggesting that most signals resulted from non-specific interactions rather than selective covalent capture. Therefore, this approach was not reliable for direct protein loss quantification. Another major focus of this dissertation was the evaluation of lysis buffer systems commonly used for cell lysis, including Reconstitution Solution (RS) and a zwitterionic buffer (ZC), to determine how buffer composition influences SP3 performance. Results showed that buffer composition strongly affected protein solubilization, binding efficiency, and peptide recovery. Some buffers produced poor recovery or higher variability, likely due to detergent interference with protein binding or digestion. In contrast, optimization of a new buffer system improved peptide recovery and reproducibility. Overall, this work provides a focused evaluation of the SP3 workflow and highlights the importance of buffer optimization, controlled processing conditions, and improved strategies for measuring protein loss to enhance the reliability of proteomics sample preparation.

Name thesis:degree_name

Master of Science

Discipline thesis:degree_discipline

Chemistry

Grantor

University of Houston

Year dc:date.issued

2026

Author dc:creator

• Islam, Shariful 1996-

Advisor dc:contributor.advisor

• Cai, Chengzhi

Committee members dc:contributor.committeemember

• Harth, Eva M.
• Comito, Robert J.
• Wang, Yuhong
• Dauwalder, Brigitte

• Sample preparation
• New optimized lysis buffer
• Protein loss
• Protein loss quantification

Language dc:language.iso

English

Handle dc:identifier.uri

https://hdl.handle.net/10657/21493

OAI identifier oai:identifier

oai:uh-ir.tdl.org:10657/21493