Microfluidic Liquid Chromatography System for Proteomic Applications and Biomarker Screening

Abstract

A microfluidic liquid chromatography (LC) system for proteomic investigations that integrates all the necessary components for stand-alone operation, i.e., pump, valve, separation column, and electrospray interface, is described in this paper. The overall size of the LC device is small enough to enable the integration of two fully functional separation systems on a 3 in. x 1 in. glass microchip. A multichannel architecture that uses electroosmotic pumping principles provides the necessary functionality for eluent propulsion and sample valving. The flow rates generated within these chips are fully consistent with the requirements of nano-LC platforms that are routinely used in proteomic applications. The microfluidic device was evaluated for the analysis of a protein digest obtained from the MCF7 breast cancer cell line. The cytosolic protein extract was processed according to a shotgun protocol, and after tryptic digestion and prefractionation using strong cation exchange chromatography (SCX), selected sample subfractions were analyzed with conventional and microfluidic LC platforms. Using similar experimental conditions, the performance of the microchip LC was comparable to that obtained with benchtop instrumentation, providing an overlap of 75% in proteins that were identified by more than two unique peptides. The microfluidic LC analysis of a protein-rich SCX fraction enabled the confident identification of 77 proteins by using conventional data filtering parameters, of 39 proteins with p < 0.001, and of 5 proteins that are known to be cancer-specific biomarkers, demonstrating thus the potential applicability of these chips for future high-throughput biomarker screening applications.

18 Coupling CE and microchip-based devices with mass spectrometry

“…Potential microchip's major advantages are: high speed for maximum ''time-to-result decrease,'' reduced sample volume and reagent consumption, integration of operational elements, disposability, portability, and high-throughput capabilities via parallel processing or automation. 313 They are engineered for sample preparation such as SPE; 314 solid-phase microextraction (SPME); 315 332 However, the latter technique is less popular since electrokinetically driven flows are more easily integrated to microstructures. It is simpler to establish a voltage drop across microchannels than pressure drops that would require miniaturized valves and pumps.…”

Microfluidic platform with mass spectrometry detection for the analysis of phosphoproteins

“…The operation of each functional element was described in detail in earlier work [51,52]. Briefly, fluidic propulsion was accomplished on both systems with the aid of the two multichannel EOF pumps (Figs.…”

“…We have previously reported a fully integrated microfluidic LC system interfaced with ESI-MS detection for proteomic analysis and biomarker screening applications [51,52]. Given the importance of phosphorylation, in this article, we have evaluated the applicability of this microfluidic platform for the analysis of phosphorylated peptides.…”

Three-Dimensional Electro-Sonic Flow Focusing Ionization Microfluidic Chip for Mass Spectrometry

“…Given the rapid development of miniaturization technology, microfluidic chips have an important function in metabolomics, proteomics, and other biochemical analyses owing to their efficient and fast separations [4,5] in integrating complex sample pretreatment functions and their automatic manipulation of small sample volumes [6][7][8][9][10]. Therefore, the coupling of microfluidic chips with MS has received considerable research interest [11], particularly the design and improvement of chip-based microfluidic ionization sources coupled with MS, which has been comprehensively reviewed by a large number of research groups [12][13][14][15].…”