Development of an automated digestion and droplet deposition microfluidic chip for MALDI-TOF MS

Abstract

An automated proteolytic digestion bioreactor and droplet deposition system was constructed with a plastic microfluidic device for off-line interfacing to matrix assisted laser desorption/ ionization time-of-flight mass spectrometry (MALDI-TOF MS). The microfluidic chips were fabricated in poly(methyl methacrylate) (PMMA), using a micromilling machine and incorporated a bioreactor, which was 100 ILm wide, 100 ILm deep, and possessed a 4 cm effective channel length (400 nL volume). The chip was operated by pressure-driven flow and mounted on a robotic fraction collector system. The PMMA bioreactor contained surface immobilized trypsin, which was covalently attached to the UV-modified PMMA surface using coupling reagents N-(3-dimethylaminopropyl)-N'-ethy1carbodiimide hydrochloride (EDC) and hydroxysulfosuccinimide (sulfo-NHS). The digested peptides were mixed with a MALDI matrix on-chip and deposited as discrete spots on MALDI targets. The bioreactor provided efficient digestion of a test protein, cytochrome c, at a flow rate of 1 ILL/min, producing a reaction time of -24 s to give adequate sequence coverage for protein identification. Other proteins were also evaluated using this solid-phase bioreactor. The efficiency of digestion was evaluated by monitoring the sequence coverage, which was 64%, 35%, 58%, and 47% for cytochrome c, Significant progress has been made toward the development of microchip-based technologies for proteomics through the integration of analytical processes into platforms that can provide rapid identification of proteins and the subsequent characterization of various post-translational modifications [1][2][3][4]. The small sample and reagent requirements, rapid analysis times, high throughput processing capabilities, and low operating costs are among the driving forces for the development of these systems [5][6][7][8]. Different microfluidic devices have been applied to specific aspects of protein processing, in particular, protein purification and separation, protein digestion, and protein identification by mass spectrometry [9].There have been a number of approaches to on-line and off-line matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) integrated to chipbased devices [10][11][12][13][14][15][16][17][18]. The primary advantage of the MALDI approach compared with electrospray ionization (ESI) when coupling to microfluidic chips is the potential for multiplexing [19]. The directional control Address reprint requests to Dr. Kermit K. Murray, Department of Chemistry, Louisiana State University, 232 Choppin Hall, Baton Rouge, LA 70802, USA. E-mail: kkmurray@!su.edu of the ESI spray can be difficult with a high-density array of spray tips. Furthermore, microfluidic chip interfaces to ESI can suffer from stability problems when sprays are started or stopped [20,21]. This limits the speed of moving from one sample to another and therefore, limits throughput.With recent advances in analytical methods for proteomics, attention has been directed toward development of effic...

Rapid and efficient proteolysis for proteomic analysis by protease‐immobilized microreactor

“…In contrast, the sequence coverage by the performed in-solution digestion (15 h at 301C) was 8% (45/607 amino acids). Lower sequence coverage was obtained for BSA, which was likely due to its stabilized tertiary structure by disulfide bonds [8,25]. The identification result by the trypsin-microreactor (5 min digestion) is even better than that by in-solution digestion that required a reaction time of 15 h.…”

“…Therefore, several enzyme-immobilized microreactors have been developed. Reported results have demonstrated that the feasibility of protein digestion using protease-immobilized on various solid supports, for example, porous monoliths [6,7], PMMA [8], glass [9], magnetic nanoparticles [10,11], and PDMS [12]. We have also developed enzyme-immobilization techniques for surface modification of microchannels and their applica- tions for the preparation of efficient enzyme microreactors [13][14][15][16].…”

Simple and Rapid Proteomic Analysis by Protease-Immobilized Microreactors

“…High sequence coverage is important to enhance the probability of identification of the protein and increase the likelihood of detection of structural variants generated by processes such as posttranslational modifications. Several methods for protease immobilization have been reported, wherein the protease, usually trypsin, has been immobilized in microchips by sol-gel encapsulation (Sakai-Kato et al, 2003;Wu et al, 2004), covalently bounded (Lee et al, 2008;Fan & Chen, 2007) and physically adsorbed onto different supports . In addition, trypsinimmobilized magnetic particles have been developted to carry out proteolysis with a short digestion time (Li et al, 2007a;.…”

A Digital Microfluidic Approach to Proteomic Sample Processing

“…There have been many inventive channel-based proteomic systems in which enzymes are immobilized in a section of the channel, such that the proteomic samples can be driven (electrokinetically or hydrodynamically) to the digestion site. 37,[171][172][173] Although such formats have favourable reaction kinetics because of the high surface-area-to-volume ratio and enzymeto-substrate ratio, such systems have typically been limited to single step processes, namely digestion. We are aware of two channel-based systems capable of carrying out a series of steps, including reduction, alkylation and digestion.…”