Gold Nanoparticle-Assisted Protein Enrichment and Electroelution for Biological Samples Containing Low Protein ConcentrationA Prelude of Gel Electrophoresis

Abstract

Protein enrichment is essential for biological samples that contain low protein concentrations, especially for proteomic studies that require sufficient quantities for subsequent MS analysis. Traditional precipitation methods, however, are limited in the sample volume and protein concentration required to cause efficient precipitations. We showed that gold nanoparticles (Au-NPs) can be easily applied to concentrate proteins from more than 15 mL of human urine, in which the total protein concentration is less than 1.4 ppm. Moreover, Au-NP-aggregated proteins can be directly applied to gel electrophoresis for Au-NP-protein dissociation followed by free protein separation as well as for the subsequent in-gel digestion and protein identification by mass spectrometry. We compared this method with trichloroacetic acid (TCA) precipitation method, one of the most common precipitation methods, and TCA method showed no enrichment effect for protein samples with large volumes (>2 mL) or with low protein concentrations (4 ppm). Therefore, Au-NP aggregation is not only a simple and efficient method for enriching a broad range of proteins, it is also particularly useful for concentrating proteins from a relatively large volume of dilute biological fluids, under which TCA method is ineffective.

Quantitative Nanoproteomics for Protein Complexes (QNanoPX) Related to Estrogen Transcriptional Action

“…Probe Fabrication-AuNPs, at a concentration of ϳ10 nM, were prepared by sodium citrate reduction following the procedures reported earlier (16). For the fabrication of the AuNP-PEG negative probe, a volume of 10 l of 500 M HS-PEG (molecular weight, 750) was added to 1 ml of synthesized AuNPs.…”

“…1C, except when centrifuged, AuNPs remain soluble and well suspended throughout the pulldown process regardless of the buffer exchange. Such advantage is superior to many other nanomaterials and is particularly useful for affinity capturing from a relatively large volume of dilute biological fluids up to several liters (16). Another unique advantage of using AuNPs as probes is the visual monitoring of the modification.…”

“…We previously demonstrated that monodispersed AuNPs are useful for concentrating proteins from a relatively large volume of dilute biological fluids by aggregation. This ability opens up new avenues of research because the traditional TCA precipitation method is ineffective under those conditions (16). Modified AuNPs have been successfully used by other groups for the detection of DNAs (17) and proteins (18) as well as for the fabrication of biosensors.…”

Recent developments of nanoparticle-based enrichment methods for mass spectrometric analysis in proteomics

“…This Au@magnetic particle-based strategy was employed to concentrate low amounts of peptide residues from the tryptic digest products of cytochrome c (10 7 M) and the trapping capacity of the Au@magnetic particles for cytochrome c was estimated to be 54 pmol/mg. In another work which also selected AuNPs as sorbents for protein enrichment, Wang et al [42] showed that gold nanoparticles could concentrate proteins from more than 15 mL of human urine, in which the total protein concentration is less than 1.4 ppm. It is noticeable that the AuNP-aggregated proteins could be directly applied to gel electrophoresis for free protein separation as well as for the subsequent in-gel digestion and protein identification by mass spectrometry.…”

Selective extraction of melamine using 11-mercaptoundecanoic acid–capped gold nanoparticles followed by capillary electrophoresis

“…Citrate-capped AuNPs were extensively used for selective enrichment of proteins [20], indoleamines [21], and polycyclic aromatic hydrocarbons [22]. Researchers successfully concentrated aminothiols from urine and serum using Tween 20-modified AuNPs [23,24].…”