Integrating Micro-Scale Separations to Matrix Assisted Laser Desorption and Ioniation Time of Flight Mass Spectrometry (MALDI-TOF-MS) for Protein Analysis

This dissertation describes the integration of micro-scale separations to matrix assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI TOF MS) for protein analysis. MALDI MS provides unsurpassed accurate mass measurements of intact bio-molecules, for example peptides and proteins, which in turn generate high molecular specificity enabling the identity, function and structure of these molecules to be characterized. However, in order to realize the full potential of MS in proteomic studies, integrated sample processing on automated and high throughput platforms is required to address the complexity, diversity and the dynamic range of proteomic analysis. The work described here contributes towards the development of automated and high throughput micro-total analysis systems (µ-TAS) for proteomics. An overview of mass spectrometry instrumentation and techniques used in protein analysis is presented to highlight the significance of the work described. Microfluidics devices can serve as automated and high throughput platforms for integrating proteomics sample processing steps such as whole cell lyses, enrichment, solubilization, denaturation, protein separations, proteolytic digestion and chromatographic separations of peptides prior to MALDI TOF MS analysis. Therefore, coupling microfluidics devices to biological mass spectrometry is the first logical step towards developing fully integrated and automated systems for protein analysis. On-line and off-line approaches for analysis from microfluidic devices are discussed. The development of a specially tailored rotating ball inlet for automated on-line MALDI MS sample introduction from an electrophoresis-based separation platform is described. Electrophoresis-based micro-scale separations of peptides on fused silica capillary and polymer-based microfluidic devices were coupled to on-line MALDI TOF MS using a rotating ball inlet. The rotating ball inlet allowed for individual technique optimization and automation thereby eliminating the need for fractionation and routine MALDI sample preparation. High throughput solid phase micro-reactors for efficient enzymatic cleavages and improved protein identification with MALDI MS in a microfluidic device were also developed for incorporation in an integrated protein analysis microfluidic system. Future work that outlines the framework and focus geared towards integrating the modules discussed in this dissertation into a functional micro-total analysis system for protein sample processing is discussed

Development of an Automated Digestion and Droplet Deposition Microfluidic Chip for MALDI-TOF MS

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 μm wide, 100 μm 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′-ethylcarbodiimide 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 μL/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, bovine serum albumin (BSA), myoglobin, and phosphorylase b, respectively. © 2008 American Society for Mass Spectrometry

Coupling a microfluidic chip to a MALDI mass spectrometer

The inherent advantages of microfluidic devices combined with the excellent sensitivity and resolution of the matrix-assisted laser desorption/ionization (MALDI) technique were discussed. The experiments were performed on a specially constructed 1 m linear time-of-flight mass spectrometer that allowed interfacing to a microfluidic chip at atmospheric pressure even though the MALDI ion source was under vacuum. The microfluidic chips were mounted on or near the source flange and held at high voltage during operation. It was observed that the chip interfaces were tested based on continuous flow MALDI, both with a frit mounted directly in the chip or with the chip effluent directed to a frit interface mounted on the source flange

Direct coupling of polymer-based microchip electrophoresis to online MALDI-MS using a rotating ball inlet

We report on the coupling of a polymer-based microfluidic chip to a MALDI-TOF MS using a rotating ball interface. The microfluidic chips were fabricated by micromilling a mold insert into a brass plate, which was then used for replicating polymer microparts via hot embossing. Assembly of the chip was accomplished by thermally annealing a cover slip to the embossed substrate to enclose the channels. The linear separation channel was 50 μm wide, 100 μm deep, and possessed an 8 cm effective length separation channel with a double-T injector (Vinj = 10 nL). The exit of the separation channel was machined to allow direct contact deposition of effluent onto a specially constructed rotating ball inlet to the mass spectrometer. Matrix addition was accomplished in-line on the surface of the ball. The coupling utilized the ball as the cathode transfer electrode to transport sample into the vacuum for desorption with a 355 nm Nd:YAG laser and analyzed on a TOF mass spectrometer. The ball was cleaned online after every rotation. The ability to couple poly(methylmethacrylate) microchip electrophoresis devices for the separation of peptides and peptide fragments produced from a protein digest with subsequent online MALDI MS detection was demonstrated. © 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Integrated microfluidic system for proteomics using mixed-scale structures and MALDI-TOF-MS

A typical sample processing pipeline for proteomics entails a series of laborious and low throughput steps that often results in long analysis times and sample loss, thereby affecting the overall efficiency and outcome of the measurement. We have assembled a polymerbased microfluidic system that includes most of the protein processing steps into a single wafer and can be interfaced to several different mass spectrometer platforms for protein identification. The system was fabricated via micro-replication technologies from polymers and possessed the ability to analyze ∼900 proteins in a single sample in an automated fashion with a total processing time \u3c1 h. © 2006 Society for Chemistry and Micro-Nano Systems

Online CE - MALDI-TOF MS using a rotating ball interface

We report on the construction and performance of a rotating ball interface for online coupling of capillary electrophoresis (CE) to matrix-assisted laser desorption ionization (BALDI) mass spectrometry with a time-of-flight (TOF) mass analyzer. The interface is based on a rotating stainless steel ball that transports samples from atmospheric pressure to the high vacuum of the mass spectrometer for desorption and ionization. The sample is deposited directly from a 50-μm-i.d. separation capillary onto the 19-mm ball that is rotating at 0.03 to 0.3 rpm. The sample is mixed online with matrix flowing from a separate 50-μm-i.d. capillary. The sample deposit dries before it is rotated past a polymer gasket and into the laser ionization region. Cleaning of the interface is accomplished using solvent-saturated felt, which cleans the ball surface after it rotates out of the ionization chamber. Online CE-MALDI is demonstrated, and the performance is evaluated with the analysis of a mixture of three peptides: [Lsy8] vasopressin, substance P, and neurotensin. The rotating ball interface to MALDI-TOF MS demonstrated mass detection limit in the high femtomole range. The interface has negligible memory effect and shows no significant electrophoretic peak broadening when operated under optimized conditions