Comprehensive Analysis of Low Molecular Weight Serum Proteome Enrichment for Mass Spectrometric Studies

Abstract

Rationale: The low molecular weight (LMW) proteins present in circulating body fluids, such as serum and plasma, hold biological significance as possible biomarkers. A major obstacle in mass spectrometry-based proteomics of serum is the presence of abundant high molecular weight proteins which mask the identification and quantitation of lower molecular weight proteins. Traditional methods involve the use of affinity resins to remove high molecular weight proteins, such as albumin and immunoglobulin G, with concomitant loss of lower molecular weight proteins. Considering the importance of depleting high molecular proteins, this paper compares an affinity resin, a gel-filter, and an acetonitrile (ACN) precipitation method to achieve successful removal of high molecular weight proteins and recovery of lower molecular weight proteins. Methods: Serum enrichment was carried out by multiple methods such as with the commercially available serum protein mini kit, ACN precipitation, and a gel filter method. Mass spectrometric runs were carried out on an AB SCIEX ESI QTOF 5600 mass spectrometer. Mass spectrometry analysis of the enriched serum obtained by ACN precipitation and gel filter method was performed for global proteome profiling. Quantitative mass spectrometry using isobaric tags for relative and absolute quantitation (iTRAQ) for ACN-precipitated enriched serum was also carried out. Results: The gel filter method, though allowing for the resolution and identification of LMW proteins, was better suited for global proteome analysis and not preferred for quantitative proteomic experiments. In contrast, enrichment by the ACN precipitation method allowed for the reproducible identification and quantitation of LMW proteins having molecular weight ≥4 kDa. Conclusions: Using only chilled ACN and centrifugation, most of the highly abundant proteins were successfully removed from the serum, while recovering a significant portion of the LMW proteome. A more rapid protocol, which is compatible with iTRAQ labeling, to achieve improved results has been elucidated, thus allowing for better screening and identification of potential biomarkers.

Surface-Enhanced Raman Analysis of Uric Acid and Hypoxanthine Analysis in Fractionated Bodily Fluids

“…Thus, the rational choice of the shape and composition, and tuning of the excitation spectral window for minimal heating and photodegradation are key elements in using nanostructured probes in biosensing [ 39 ]. The intrinsic hotspots of gold nano stars gave quite different spectral peaks compared to assign bands in SERS spectra of chemicals, serum and plasma in literature [ 3 , 12 , 19 ]. The current study shows that the limits of detection (LOD) for uric acid are consistently higher than those for hypoxanthine, across all biofluids, using the same SERS approach.…”

“…However, the analysis of bodily fluids is considered challenging, to a large degree due to the high dynamic concentration range of constituent protein/peptide species. While SERS, using colloidal gold or silver nanoparticles, has been used extensively to characterise blood fluids for diagnostic applications [ 17 , 18 , 19 ], it has been pointed out that the NP surfaces may have a more specific affinity for some biomolecules present in the complex millieu of the biofluid than others, and that therefore the measured SERS spectrum can be dominated by them [ 20 ]. While this may limit the sensitivity of the technique for some applications, uric acid and hypoxanthine have been identified as molecules with a particular affinity to gold and silver NP surfaces, and therefore the analysis should be particularly sensitive to any diagnostic procedure based on variations in the concentrations of these molecules [ 21 ].…”

Simultaneous Quantification of Apolipoprotein C-III O-Glycoforms by Protein-MRM

“…For protein-MRM, APOC-III protein enrichment was optimized using various reagents (e.g., ACN or acids) at different conditions, including different concentrations and temperatures. ,− Typically, 10 μL of human serum was diluted with 20 μL of distilled water, and chilled ACN was added up to a 60% concentration; the mixture was mixed gently and incubated for 30 min at room temperature. Then, protein pellets were removed by centrifugation (Eppendorf, 5424R) at 4 °C, 21,000 g for 10 min, and small particles in the supernatant were removed by centrifuge filtration with an in-house C18 tip (Empore 47 mm C18 extraction disks, 3 M, 2215-47MM-6).…”

Quantitative Serum Proteomics Reveals The Predictive and Prognostic Potential of Clusterin and Gelsolin in Head and Neck Squamous Cell Carcinoma Treated With Radiotherapy

“…Serum enrichment was carried out as per the protocol standardized by our group [23]. Brie y, 10mg of serum, from a total protein concentration of 60-70 mg/ml, was directly precipitated using chilled acetonitrile (ACN) in 1:1 ratio.…”