Protein digestion and phosphopeptide enrichment on a glass microchip
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.
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A selective metabolite array for the detection of phosphometabolites
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