Elsevier

Analytica Chimica Acta

Volume 564, Issue 1, 30 March 2006, Pages 116-122
Analytica Chimica Acta

Protein digestion and phosphopeptide enrichment on a glass microchip

https://doi.org/10.1016/j.aca.2005.11.003Get rights and content

Abstract

This work describes an integrated glass microdevice for proteomics, which directly couples proteolysis with affinity selection. Initial results with standard phosphopeptide fragments from β-casein in peptide mixtures showed selective capture of the phosphorylated fragments using immobilized metal affinity chromatography (IMAC) beads packed into a microchannel. Complete selectivity was seen with angiotensin, with capture of only the phosphorylated form. On-chip proteolysis, using immobilized trypsin beads packed into a separate channel, was directly coupled to the phosphopeptide capture and the integrated devices evaluated using β-casein. Captured and eluted fragments were analyzed using both capillary electrophoresis (CE) and capillary liquid chromatography/mass spectrometry (cLC/MS). The results show selective capture of only phosphopeptide fragments, but incomplete digestion of the protein was apparent from multiple peaks in the CE separations. The MS analysis indicated a capture bias on the IMAC column for the tetraphosphorylated peptide fragment over the monophosphorylated fragment. Application to digestion and capture of a serum fraction showed capture of material; however, non-specific binding was evident. Additional work will be required to fully optimize this system, but this work represents a novel sample preparation method, incorporating protein digestion on-line with affinity capture for proteomic applications.

Introduction

The development of lab-on-a-chip or micro-total analysis systems (μ-TAS) has gained a significant amount of attention over the past decade. Microchip technology, originally shown to expedite electrophoresis separations, offers the additional promise of allowing integrated sample preparation in micron-sized chambers or channels fabricated in glass or polymer analytical devices [1], [2]. In addition to the potential for automating sample preparation and analysis, scaling down the analytical processes also offers faster total speed and potential savings on reagent costs. With respect to application of these devices for proteomics, the method of detection determines what sample preparation steps are required in the analysis [1]. For proteins, mass spectrometry (MS) is most often utilized for detection because it can provide accurate information for protein identification in a sample as well as provide information about post-translational modifications present. Toward this end, a large number of papers have illustrated integration of mass spectrometry detection directly from the end of the microdevices [3], [4], [5], [6], [7]. To exploit the potential capabilities of sample preparation microchips for protein analysis using MS, efforts have been directed toward demonstrating on-chip digestion, and separations that could be integrated with MS detection on a single device [8], [9], [10], [11], [12], [13], [14].

With the global proteomic effort gaining momentum, methods that facilitate understanding the post-translational modification (PTM) of proteins become increasingly important. Protein phosphorylation, for example, is known to play an important role in a number of processes including signal transduction [15], [16]. Knowledge of the particular residue that is phosphorylated can provide insight into a signaling pathway via an understanding of how the protein's activity is regulated and which enzymes are responsible for the regulation. A number of methods have successfully been developed to enrich phosphopeptides for analysis [17], [18], [19], the most amenable of which to microdevices is immobilized metal ion affinity chromatrography (IMAC). In IMAC, an immobilized metal (Fe3+ or Ga3+) on a solid support (such as Sepharose) can selectively retain and pre-concentrate phosphorylated proteins and peptides; this method has been successfully applied for phosphopeptide enrichment by a number of groups [20], [21], [22]. A typical procedure for phosphopeptide enrichment with IMAC incorporates digestion of the protein with trypsin to generate peptide fragments, normally performed overnight.

The present work describes a new application of microdevices for combined digestion of proteins followed by on-line phosphopeptide capture with IMAC on a single glass microdevice. Agarose beads containing immobilized trypsin were packed into one channel, with agarose beads containing ferric ion packed into a subsequent channel. The frit for solid phase (beads) packing was made simply by reducing the diameter of the channel in the microchip. In a similar manner, this microdevice was also used for enrichment of glycopeptides through lectin affinity capture. Both of these methods could be directly integrated with MS detection from the same microdevice through the proper microchip interface.

Section snippets

Materials

Trypsin, β-casein, ovalbumin and cytochrome c, purchased from Sigma–Aldrich (St. Louis, MO), were each dissolved in 0.1 M NH4HCO3 (pH 8.0) at a concentration of 1 mg/mL. Immobilized trypsin beads, agarose beads with immobilized iminodiacetic acid, and agarose beads with immobilized Concanavalin A, were all purchased from Pierce (Rockford, IL). Acetic acid, FeCl3, and sodium phosphate were purchased from Fisher. β-casein phosphopeptide standards, phospho-angiotensin standard, and ammonium

Phosphopeptide enrichment using the IMAC microchip

To test the specificity of IMAC enrichment on a microchip, standard phosphopeptides were mixed with non-phosphopeptides and loaded on IMAC microchips. In the first experiments, 20 μg of peptides from cytochrome c digested with trypsin was mixed with 16 μg of phospho-angiotensin and loaded on an IMAC microchip. The captured peptides were eluted from the device and the eluent was separated by CE. Electropherogram a in Fig. 1 shows the electrophoretic separation of the cytochrome c digest mixed with

Discussion

Mass spectrometry is the preferred detection method in proteomics because of the sensitivity and the ability to identify specific proteins present in a sample. Normal sample processing for MS detection requires trypsin digestion of sample proteins, with affinity purifications sometimes utilized to identify specific peptide fragments, such as those with post-translational modifications. Microdevices have been utilized for both interfacing with the MS detection, and for the trypsin digestion part

Conclusion

Phosphopeptide capture from standard solutions and protein digests has been achieved on a simple microdevice using IMAC beads packed into a microfluidic channel. Replacement of the IMAC beads with immobilized lectin beads allowed capture of a glycoprotein in this same device. The phosphopeptide capture method has been integrated with on-chip trypsin digestion, requiring additional flow through a side inlet to decrease the pH of the digestion solution for effective binding to the IMAC beads.

Acknowledgements

The Authors wish to thank Katie Horsman and Deb Lannigan for their help with the serum fraction analysis, and An Chi for MS analysis of the treated serum fraction.

References (24)

  • R.D. Oleschuk et al.

    Trends Anal. Chem.

    (2000)
  • L. Korecka et al.

    J. Chromatogr. B: Anal. Tech. Biomed. Life Sci.

    (2004)
  • M.J. Hubbard et al.

    Trends Biochem. Sci.

    (1993)
  • W. Zhou et al.

    Am. Soc. Mass Spectrom.

    (2000)
  • J.P. Ferrance et al.
  • J.P. Landers

    Anal. Chem.

    (2003)
  • G.E. Yue et al.

    Lab. Chip

    (2005)
  • S. Ssenyange et al.

    Anal. Chem.

    (2004)
  • R.S. Ramsey et al.

    Anal. Chem.

    (1997)
  • B. Zhang et al.

    Anal. Chem.

    (2000)
  • I.M. Lazar et al.

    Anal. Chem.

    (2001)
  • J. Gao et al.

    Anal. Chem.

    (2001)
  • Cited by (33)

    • Solid supports for extraction and preconcentration of proteins and peptides in microfluidic devices: A review

      2017, Analytica Chimica Acta
      Citation Excerpt :

      Several bead-based processes, requiring significantly different chemistry, can be integrated into a single device. For instance, Yue and coworkers coupled protein digestion on trypsin-grafted beads with subsequent selective phosphopeptide capturing on immobilized metal-affinity chromatography (IMAC) bed [28]. The enzymatic cleaving and digest enrichment was performed in the same channel under continuous sample flow with beds connected in series.

    • A selective metabolite array for the detection of phosphometabolites

      2012, Analytica Chimica Acta
      Citation Excerpt :

      This method has been widely used in proteomics for low abundant phosphopeptide and phosphoprotein separation and enrichment [19]. Metal ion immobilisation on to chromatography support [20], planar substrates [21], and more recently MALDI plate [22], for direct MS analysis have been explored to minimise sample loss. Various planar surface modification approaches that have been used include self-assembled monolayers of ligands [23], layer-by-layer deposition of polyelectrolytes for metal binding [24], metal immobilisation to polymer-coated substrates [25,26], and electrospray deposition of metal oxides [27].

    • The design of microfluidic affinity chromatography systems for the separation of bioanalytes

      2012, Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences
      Citation Excerpt :

      Integration of the different separation, processing and analysis steps into one lab-on-a-chip device would be highly desirable and would offer an approach requiring limited sample handling [3]. Such a lab-on-a-chip format offers the possibility to have many parallel analysis channels, each containing many sequential steps such as enzymatic digestion, multiple separation steps and connection to in-line detection methods [4,5]. So far some of the necessary component elements, needed for integration within lab-on-a-chip devices for the multiplexed analysis of complex protein mixtures, have been created [6].

    View all citing articles on Scopus
    View full text