Derivatized Nanoparticle Coated Capillaries for Purification and Micro-extraction of Proteins and Peptides

Abstract

Various methods are used to enrich or purify a protein of interest from other proteins and components in a crude cell lysate or other sample. One of the most powerful methods is affinity purification, also called affinity chromatography, whereby the proteins of interest are purified by virtue of their specific binding properties to an immobilized ligand. Affinity purification is becoming more widely used for exploring post-translation modifications and protein-protein interactions, especially with a view toward developing new general tag systems and strategies of chemical derivatization on peptides for affinity selection. Our work was aimed to immobilize proteins or ligands for affinity purification of antibodies, fusion-tagged proteins and other proteins and peptides. Selected proteins or peptides are efficiently extracted and enriched using chemically derivatized walls of a fused silica capillary column. In this paper, we present an open tubular capillary, where the inner wall of a fused silica capillary was derivatized by covalent binding of modified polystyrene latex particles. The capillaries were derivatized with iminodiacetic acid and loaded with Fe3+ or Ni2+ for the purification and enrichment of phosphopeptides or His-tagged proteins, respectively. The latex coated capillaries have been successfully applied to enrich phosphopeptides from beta-casein tryptic digest and ovalbumin tryptic digest at a micro volume scale with recoveries ranging from 92 to 95%. The capillaries have been eluted under conditions compatible with MALDI-MS without any prior desalting step. In another approach, concanavalin A (Con A) or Protein G were immobilized on the epoxy modified latex on the inner wall of the fused silica capillary for the purification of glycoproteins and immunoglobulin, respectively. The design of the capillary and the protocols used for purification permits the direct detection of eluted proteins and peptides with gel electrophoresis or with mass spectrometry. The elution volumes are passed as discrete segments of few microliters over the inner surface of the open-tube capillary, achieving enrichment factors of more than 20-fold from starting samples.

Surface-area expansion with monolithic open tubular columns

“…The ratio of the surface area to the volume plays a key role regarding the capacity of such PLOT columns, whilst recent progress in non-material science focused attention on various kinds of nanoobject. To illustrate, latex NPs have been immobilized by reaction of introduced epoxy groups on the NPs with (3-aminopropyl) trimethoxysilane-modified silica surfaces and then further used for the microextraction of peptides [53]. Similarly, polystyrene NPs modified with BSA have been used for the enantioseparation of R-and S-tryptophan by CEC [54], whilst C60-fullerene has been covalently bonded via a "photo click" coupling reaction with the derived stationary phase evaluated for the separation of polycyclic aromatic hydrocarbons [55].…”

Immobilized metal affinity chromatography using open tubular capillary for phosphoprotein analysis: Comparison between polymer brush coating and surface functionalization

“…The method used to attach iminodiacetic acid onto the surface of a fused silica capillary is a modified version of the one used by Bakry et al to prepare latex-coated capillary IMAC columns [46]. IDA (0.5 g) was dissolved in a 25 mL Na 2 CO 3 solution (2 M) containing 250 mg NaCl.…”

In Situ Measurement of Polymer Layer Thickness in Porous Layer Open Tubular (PLOT) Columns Using Optical Absorbance in the Near-IR Range

“…Porous layer open tubular (PLOT) columns (see Figure 1) are a type of open tubular column used in a variety of chromatography applications, and although they find some use in liquid chromatography (LC) [1][2][3][4][5][6][7][8][9], capillary electrochromatography [10][11][12][13][14], and micro solid phase extraction (µ-SPE) [15,16], their main area of application is in gas chromatography (GC) [17,18]. Typically, such columns are fabricated through the immobilisation of spherical particles onto the inner wall of the capillary.…”