Interfacing capillary gel microfluidic chips with infrared laser desorption mass spectrometry

Abstract

We report on the fabrication and performance of a gel microfluidic chip interfaced to laser desorption/ionization (LDI) mass spectrometry with a time-of-flight mass analyzer. The chip was fabricated from poly(methylmethacrylate) with a poly(dimethyl siloxane) cover. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed in the channel of the microfluidic chip. After electrophoresis, the cover was removed and either the PDMS chip or the PMMA cover was mounted in a modified MALDI ion source for analysis. Ions were formed by irradiating the channel with 2.95 m radiation from a pulsed optical parametric oscillator (OPO), which is coincident with IR absorption by N-H and O-H stretch of the gel components. No matrix was added. The microfluidic chip design allowed a decrease in the volume of material required for analysis over conventional gel slabs, thus enabling improvement in the detection limit to a pmol level, a three orders of magnitude improvement over previous studies in which desorption was achieved from an excised section of a conventional gel. ( T he rapid expansion of the field of proteomics has created an increased demand for selective, sensitive, and high-throughput methods of analysis. Soft ionization methods, such as matrix-assisted desorption/ionization (MALDI) [1,2] and electrospray (ESI) [3,4] are suitable for achieving the goal of identification and structural determination of proteins. Mass spectrometry (MS) has become the primary analysis method in this area because of its high throughput, sensitivity, and high mass accuracy. However, the large number and broad concentration range of the proteins present in the expressed proteome of a typical organism requires that one or more fractionation or separation steps be performed on the sample before mass spectrometry analysis.One of the most commonly utilized techniques for protein separation is sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) [5][6][7][8][9]. The molecular weight of a protein is determined by the migration distance after electrophoretic separation together with several marker proteins of known molecular weights that bracket the protein of interest. Currently, most proteome analyses are based on the separation of complex protein mixtures by 1D or 2D gel electrophoresis, followed by mass spectrometric analysis of proteolytic digests of gel spots to identify individual proteins [10 -13]. However, this protocol requires intermediate sample processing steps such as extraction of the protein from the gel [13], blotting the protein onto a membrane [14], or electroelution [15], which introduces a number of laborious and time-consuming manual steps that are difficult to automate.Various efforts have been directed toward direct analysis of gel and planar chromatography separations by laser desorption ionization (LDI) mass spectrometry. The mass spectrometric analysis of peptides and proteins by UV-MALDI directly from polyacrylamide gels has been reported previously [16]. Here, ultrathin gels (less than 10 m) ...

Infrared Laser Ablation Sample Transfer for MALDI and Electrospray

“…The volumetric energy deposition with a mid-IR laser is comparable to that of UV lasers, but the depth of laser penetration is an order of magnitude larger [22]. The greater penetration depth of IR lasers can be an advantage in removing deeply embedded materials such as biomolecules in polyacrylimide gels [23,24] and in tissue samples [25]. Another advantage of mid-IR lasers for ablation of material is that the characteristics of the ablation plume can be widely varied simply by changing the IR laser wavelength in a wavelength tunable system [26,27].…”

Lab‐on‐a‐plate: Extending the functionality of MALDI‐MS and LDI‐MS targets

“…Another important improvement is the matrix-free IR-LDI analysis of peptides and proteins from a polyacrylamide gel (Xu et al, 2004a). Furthermore, matrix-free IR-LDI of polypeptides and proteins could also be performed after gel electrophoretic separation in a microfluidic chip (Xu, Little, & Murray, 2006). Surprisingly, the literature on PAGE-MALDI is considerably scarcer than that on TLC-MALDI, although PAGE is certainly much more widespread as a bioanalytical separation method than TLC.…”

Recent progress in microchip electrophoresis–mass spectrometry

“…Especially in ESI-MS, the nonvolatile additives cause low ionization efficiency and lower detectability of analytes. To overcome this limitation, the applications of LDI to MCE-MS have been investigated [52][53][54][55][56][57]. In the first MCE-LDI-MS report, the MCE separation with a BGS containing conventional matrix component (2,5-dihydroxybenzoic acid) in MALDI is performed in an openaccess channel fabricated on a glass microchip, and the chips are transferred to a MALDI source after the solvents is evaporated, i.e., the channel yields a MALDI sample complete with matrix and ready for analysis [52].…”