Application of Microfluidic Devices to Proteomics Research

Abstract

This report describes an integrated and modular microsystem providing rapid analyses of trace-level tryptic digests for proteomics applications. This microsystem includes an autosampler, a microfabricated device comprising a large channel (2.4 l total volume), an array of separation channels, together with a low dead volume enabling the interface to nanoelectrospray mass spectrometry. The large channel of this microfluidic device provides a convenient platform to integrate C 18 reverse phase packing or other type of affinity media such as immobilized antibodies or immobilized metal affinity chromatography beads thus enabling affinity selection of target peptides prior to electrophoretic separation and mass spectrometry analyses on a quadrupole/time-of-flight instrument. Sequential injection, preconcentration, and separation of peptide standards and tryptic digests are achieved with a throughput of up to 12 samples/per h and a concentration detection limit of ϳ5 nM (25 fmol on chip). Replicate injections of peptide mixtures indicated that reproducibility of migration time was 1.2-1.8%, whereas relative standard deviation ranging from 9.2 to 11.8% are observed on peak heights. The application of this device for trace-level protein identification is demonstrated for two-dimensional gel spots obtained from extracts of human prostatic cancer cells (LNCap) using both peptide mass-fingerprint data base searching and on-line tandem mass spectrometry. Enrichment of target peptides prior to mass spectral analyses is achieved using c-myc-specific antibodies immobilized on protein G-Sepharose beads and facilitates the identification of antigenic peptides spiked at a level of 20 ng/ml in human plasma. Affinity selection is also demonstrated for gelisolated protein bands where tryptic phosphopeptides are captured on immobilized metal affinity chromatography beads and subsequently separated and characterized on this microfluidic system. Molecular & Cellular Proteomics 1:157-168, 2002.Proteomics research entails the global characterization of proteins expressed in cells under defined conditions. Such studies are particularly important in view of the conflicting evidence regarding the correlation between the abundance of expressed proteins and gene-expression levels obtained from mRNA microarrays (1, 2). The monitoring of protein expression profiles remains a very challenging task because of the wide dynamic range of expressed proteins and the variability of gene products (splicing variants, N-and C-terminal truncations, co-and post-translational modifications, etc.), which may change between and within the tissues of an organism (3). The traditional approach to isolating and characterizing proteins from biological samples has been separation by twodimensional gel electrophoresis (2-D 1 gel), followed by identification of protein spots using sensitive mass spectrometry techniques and data base searching (3-5). An alternate approach to 2-D gel includes a comprehensive chromatographic separation of the proteolytic fragments derive...

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

“…ignificant 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].…”

A Comparative Study of Protein Profiling by Proteomic Analysis in Camptothecin-Resistant PC3 and Camptothecin-Sensitive LNCaP Human Prostate Cancer Cells

“…applied for the detection of other characteristic proteins in tissue and serum from prostate cancer patients [1][2][3][4][5] , prostasomes (secretory vesicles in epithelial cells) in seminal fl uid [6] and the prostate cancer cell line LNCaP (cellular proteins and proteins released in response to androgen) [7][8][9][10] . Recently, a mitochondrial proteome analysis using a new type of protein lysate array has been applied for detecting any altered energy metabolism in prostate cancer tissue samples [11] .…”

Microfluidic systems in proteomics

“…First the packing material must be flown and trapped into the microchannel; Li et al [39] used a first plug of 40 mm C18 beads, to make a filter at the junction of a large 800 mm wide, 150 mm deep and 22 mm long channel and a small 10 mm deep, 30 mm wide injection channel. The large channel was then packed with 5 mm C18 beads, resulting in a surfaceto-volume ratio around 10 7 m 21 (estimated from authors' data and Waters' technical information).…”