Evaluation of Sample Preparation Strategies for Human Milk and Plasma Proteomics

Abstract

Sample preparation is the most critical step in proteomics as it directly affects the subset of proteins and peptides that can be reliably identified and quantified. Although a variety of efficient and reproducible sample preparation strategies have been developed, their applicability and efficacy depends much on the biological sample. Here, three approaches were evaluated for the human milk and plasma proteomes. Protein extracts were digested either in an ultrafiltration unit (filter-aided sample preparation, FASP) or in-solution (ISD). ISD samples were desalted by solid-phase extraction prior to nRPC-ESI-MS/MS. Additionally, milk and plasma samples were directly digested by FASP without prior protein precipitation. Each strategy provided inherent advantages and disadvantages for milk and plasma. FASP appeared to be the most time efficient procedure with a low miscleavage rate when used for a biological sample aliquot, but quantitation was less reproducible. A prior protein precipitation step improved the quantitation by FASP due to significantly higher peak areas for plasma and a much better reproducibility for milk. Moreover, the miscleavage rate for milk, the identification rate for plasma, and the carbamidomethylation efficiency were improved. In contrast, ISD of both milk and plasma resulted in higher miscleavage rates and is therefore less suitable for targeted proteomics.

Comparative proteomics of human milk casein fraction collected from women of Korean and Han ethnic groups in China

“…Enzymatic hydrolysis of protein samples was conducted using FASP according to a previous study ( 15 ), which seems to be the most effective approach for biological sample aliquots owing to the low miscleavage rate ( 16 ). To each protein sample (100 μg) 8 M urea was added to obtain a final volume of 200 μL, and then DL-dithiothreitol was added until a final concentration of 10 mM; this mixture was incubated at 56°C for 30 min to destroy the intramolecular and intermolecular disulfide bonds.…”

Establishing Quality Control Metrics for Large-Scale Plasma Proteomic Sample Preparation

“…For protein depletion, it has been reported that MARS-14 columns do not efficiently capture targeted proteins. 27 Therefore, a 90% MARS-14 DE was chosen as the desired specification because it is dependent on protein concentrations obtained from the BCA assay and not target identification that would require an LC−MS/MS check. If the results were OOS, participant plasma depletions were paused, and another QC std aliquot was depleted for confirmation.…”

“…Ideally, sample preparation workflows should be designed to minimize sample handling and operator-generated biases. However, sample preparation can be tedious, error-prone, and susceptible to reproducibility issues. − Automation with robotic liquid handlers relieves the burden of sample preparation while also reducing variability between operators and samples . These handlers are also embraced for reducing the cost of routine biomarker discovery and development .…”

Lysis buffer selection guidance for mass spectrometry-based global proteomics including studies on the intersection of signal transduction and metabolism

“…In this manner, SP3 is recognized as a more versatile and efficient approach to sample preparation, enhancing the adaptability of protocols and facilitating the use of reagents previously considered off-limits for MS-based proteomics. Despite previous studies successfully comparing widely used MS sample preparation methods (e.g., classical ISD and FASP) [12][13][14], there has been a notable gap in research, and a systematic evaluation of different lysis buffer reagents using a single sample preparation strategy has been missing. Furthermore, lysates can be utilized to enrich for interaction partners before the analysis by MS.…”