Multifunctional protein processing chip with integrated digestion, solid‐phase extraction, separation and electrospray

Abstract

We describe a microfluidic device in which integrated tryptic digestion, SPE, CE separation and electrospray ionization for MS are performed. The chip comprised of 10 × 30 μm channels for CE, and two serially connected 150 μm deep, 800 μm wide channels packed with 40 to 60 μm diameter beads, loaded with either immobilized trypsin, reversed-phase packing or both. On-chip digestion of cytochrome c using the trypsin bed showed complete consumption of the protein in 3 min, in contrast to the 2 h required for conventional solution phase tryptic digestion. SPE of 0.25 μg/mL solutions of the peptides leu-enkephalin, angiotensin II and LHRH gave concentration enhancements in the range of 4.4-12, for a ten times nominal volume ratio. A 100 nM cytochrome c sample concentrated 13.3 times on-chip gave a sequence coverage of 85.6%, with recovery values ranging from 41.2 to 106%. The same sample run without SPE showed only five fragment peaks and a sequence coverage of 41.3%. When both on-chip digestion and SPE (13.3 volume ratio concentration enhancement) were performed on 200 nM cytochrome c samples, a sequence coverage of 76.0% and recovery values of 21-105% were observed. Performing on-chip digestion alone on the same sample gave only one significant fragment peak. The above digestion/peptide concentration step was compared to on-chip protein concentration by SPE followed by on-chip digestion with solution phase trypsin. Both procedures gave similar recovery results; however, much larger trypsin autodigestion interference in the latter approach was apparent.

Microfluidic chips with reversed-phase monoliths for solid phase extraction and on-chip labeling

“…The most common SPE modes in microfluidics are affinity [4,17,18] and reversed-phase [6,19,20]. Affinity SPE in microchips has been used to extract and quantify four cancer biomarkers in blood [4], to preconcentrate and purify PCR products [17], and to extract thiazole orange-conjugated adenosine monophosphate [18].…”

“…Reversed-phase columns are useful in the extraction of non-polar to moderately polar compounds. Silica-based materials are common reversed-phase SPE supports, having been used for the extraction of parabens and fluorescent dyes [6], the preconcentration of peptides and cytochrome c [19], and the concentration and separation of Rhodamine 123 and fluorescein isothiocyanate (FITC)-labeled ephedrine [20]. Monolithic columns are seeing increased usage because they can be easily prepared on-chip without the need for retaining structures like frits [5,21], and the porosity and surface area can be tuned by varying the monomer/porogen composition [22].…”

Microfluidics-to-mass spectrometry: A review of coupling methods and applications

“…In a study by the Harrison group [35], a multifunctional microfluidic device in which integrated trypsin digestion, SPE, electrophoretic separation, and ESI-MS was demonstrated (Figure 10). Two packed beds filled with 40–60 μm diameter beads, loaded with either immobilized trypsin, reversed-phase packing, or both, were used as the digestion and SPE column, respectively.…”

“…Both beds were 150 μm deep and 800 μm wide loaded with 40–60 μm diameter beads. Adapted with permission from [35]. Copyright (2010) John Wiley and Sons.…”

Microchip Emitter for Solid-Phase Extraction–Gradient Elution–Mass Spectrometry

“…20,22 As the eluate is separated by electrophoresis, SPE serves only for the enrichment of the analyte and elimination of the impurities. The integration of a multistep elution from SPE sorbents packed inside the separation capillary in CE-MS/MS 23 provides an additional possibility to increase the resolving power of the system.…”