A microfabricated silicon platform with 60 microfluidic chips for rapid mass spectrometric analysis (original) (raw)
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Analytical Chemistry, 2012
The potential benefits of ultra-low flow electrospray ionization (ESI) for the analysis of phosphopeptides in proteomics was investigated. First, the relative flow dependent ionization efficiency of nonphosphorylated vs multiplyphosphorylated peptides was characterized by infusion of a five synthetic peptide mix with zero to four phophorylation sites at flow rates ranging from 4.5 to 500 nL/min. Most importantly, similar to what was found earlier by Schmidt et al., it has been verified that at flow rates below 20 nL/min the relative peak intensities for the various peptides show a trend toward an equimolar response, which would be highly beneficial in phosphoproteomic analysis. As the technology to achieve liquid chromatography separation at flow rates below 20 nL/min is not readily available, a sheathless capillary electrophoresis−electrospray ionization-mass spectrometry (CE− ESI-MS) strategy based on the use of a neutrally coated separation capillary was used to develop an analytical strategy at flow rates as low as 6.6 nL/min. An in-line preconcentration technique, namely, transient isotachophoresis (t-ITP), to achieve efficient separation while using larger volume injections (37% of capillary thus 250 nL) was incorporated to achieve even greater sample concentration sensitivities. The developed t-ITP-ESI-MS strategy was then used in a direct comparison with nano-LC−MS for the detection of phosphopeptides. The comparison showed significantly improved phosphopeptide sensitivity in equal sample load and equal sample concentration conditions for CE−MS while providing complementary data to LC−MS, demonstrating the potential of ultra-low flow ESI for the analysis of phosphopeptides in liquid based separation techniques.
Analytical Chemistry, 2004
A compact disk (CD)-based microfluidic method for selective detection of phosphopeptides by mass spectrometry is described. It combines immobilized metal affinity chromatography (IMAC) and enzymatic dephosphorylation. Phosphoproteins are digested with trypsin and processed on the CD using nanoliter scale IMAC with and without subsequent in situ alkaline phosphatase treatment. This is followed by on-CD matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. Dephosphorylation of the IMAC-enriched peptides allows selective phosphopeptide detection based on the differential mass maps generated (mass shifts of 80 Da or multiples of 80 Da). The CD contains 96 microstructures, each with a 16 nL IMAC microfluidic column. Movement of liquid is controlled by differential spinning of the disk. Up to 48 samples are distributed onto the CD in two equal sets. One set is for phosphopeptide enrichment only, the other for identical phosphopeptide enrichment but combined with in situ dephosphorylation. Peptides are eluted from the columns directly into MALDI target areas, still on the CD, using a solvent containing the MALDI matrix. After crystallization, the CD is inserted into a MALDI mass spectrometer for analysis down to the femtomole level. The average success rate in phosphopeptide detection is over 90%. Applied to noncharacterized samples, the method identified two novel phosphorylation sites, Thr 735 and Ser 737, in the ligand-binding domain of the human mineralocorticoid receptor.
Chip-based microfluidic devices coupled with electrospray ionization-mass spectrometry
ELECTROPHORESIS, 2005
Chip-based microfluidic devices coupled with electrospray ionization-mass spectrometry We present the current status of the development of microfluidic devices fabricated on different substrates for coupling with electrospray ionization-mass spectrometry (ESI-MS). Until now, much success has been gained in fabricating the ESI chips, which show better performances due to miniaturization when compared with traditional methods. Integration of multiple steps for sample preparation and ESI sample introduction, however, remains a great challenge. This review covers the main technical development of electrospray device that were published from 1997 to 2004. This article does not attempt to be exclusive. Instead, it focuses on the publications that illustrated the breath of the development and applications of microchip devices for MS-based analysis.
Analytical and Bioanalytical Chemistry, 2012
Quantitative detection of phosphorylation levels is challenging and requires an expertise in both stable isotope labeling as well as enrichment of phosphorylated peptides. Recently, a microfluidic device incorporating a nanoliter flow rate reversed phase column as well as a titania (TiO 2 ) enrichment column was released. This HPLC phosphochip allows excellent recovery and separation of phosphorylated peptides in a robust and reproducible manner with little user intervention. In this work, we have extended the abilities of this chip by defining the conditions required for on-chip stable isotope dimethyl labeling allowing for automated quantitation. The resulting approach will make quantitative phosphoproteomics more accessible.
Microfabricated Dual Sprayer for On-Line Mass Tagging of Phosphopeptides
Analytical Chemistry, 2008
, called tag) were performed with a dual-channel microsprayer in electrospray ionization mass spectrometry. The reaction is first studied ex situ and analyzed with a commercial electrospray source. In situ reactions (i.e., inside the Taylor cone) were achieved with a dual-channel microsprayer both with the tag synthesized chemically before the experiments and with the tag electrogenerated by in situ oxidation of a zinc electrode, also used to apply the electrospray current. The device consists of a polyimide microchip with two microchannels (20 µm × 50 µm × 1 cm) etched on each side of the structure and connecting only at the tip of the microchip. We demonstrate here that mixing two solutions with different physicochemical properties inside the Taylor cone can be used to selectively tag target molecules. Post-translational modifications (PTMs) appear in the last step in the maturation of proteins, such as phosphorylation, glycosylation, or alkylation. Phosphorylation of proteins is the most studied PTM in living systems. It is a reversible modification adjusting folding and function of proteins, e.g., enzymatic activities or regulating protein localization, and it is involved in many crucial cell functions such as signal transduction, metabolic maintenance, or cell division. 1 Four types of phosphorylation are known, 2 all involving the addition of a phosphate group on an amino acid. O-phosphates are present on serine, tyrosine, and threonine residues and are the most common. N-, S-, and acyl-phosphorylations can mostly occur on histidine or lysine, cysteine, and aspartic or glutamic acid, respectively. Nowadays, all these PTMs are mostly well understood. However, the analysis of phosphoproteins and phosphopeptides remains a key challenge of proteome research. Indeed, the abundance of phosphoproteins is very low (1-2% of native protein), and some special sample preparation and sample preconcentration are required to isolate phosphocompounds. 3 The detection of phosphoproteins can be performed in 2D gels by phospho-specific stains or by western blotting techniques. These methods are quantitative and can detect all kinds of phosphorylation. Nevertheless, comigrating proteins can falsify the results; then a distinct stained protein plot is not necessarily representative of a unique protein and additional measurements such as mass spectrometry (MS) have to be performed. Moreover, the detection of phosphorylated proteins or peptides by MS is difficult due to their low ionization efficiencies. Indeed, in positive ionization mode, the ionization of these species depends of the number of basic residues, which contribute to the global net charge of the peptide or protein. Phosphorylation increases the acidity of peptides and hence decreases the protonation in a solution at pH values typically used in MS. Chemical modifications 4-6 or phosphopeptide enrichment strategies have thus been developed. Liquid chromatography (LC) is a method of choice for sample preconcentration and coupling with MS. The isolation of phosphopeptides in affinity chromatography can be done either by retention on immobilized metal ion affinity chromatography 7-10 or by immobilization in TiO 2 columns. 11,12 Electrostatic interactions between the stationary phase and the phosphopeptides allow the separation of the different compounds. To improve the detection by MS, several procedures have been developed such as the identification by metastable decomposition with a matrix-assisted laser desorption/ionization (MALDI) MS 13 or by chemical modification, [14][15][16] or such as the preconcentration with Fe 3+ -Wollscheid, B.; O'Brien, R.; Eng, J. K.; Li, X. J.; Bodenmiller, B.; Watts, J. D.; Hood, L.; Aebersold, R. Nat. Methods 2005, 2, 591-598. (5) Thompson, A. J.; Hart, S. R.; Franz, C.; Barnouin, K.; Ridley, A.; Cramer, R.
Sensors and Actuators B: Chemical, 2008
This article presents the development and performance of new silicon microfluidic devices, called Espray chips, integrating both a reversed-phase separation column and a nano-electrospray emitter. The microchips are made according to standard silicon microtechnology procedures including photolithography, deep reactive ion etching and molecular bonding. The separation column is a perfectly ordered 2-dimensional array of squared micropillars, directly etched in the silicon substrate, and the electrospray emitter is a planar nib-like nanotip. Two chemical procedures for the separation column reversed-phase coating were tested: (i) a liquid phase "chip by chip" process with a C18-alkylated silane and (ii) a vapour phase collective process with a C10-perfluorated silane. Analyses of standard tryptic digests of cytochrome c in hydrodynamic pumping mode have demonstrated good quality spray and effective separation performance of these microdevices with a higher retention capacity for C10-perfluorated coating. These new microchips, which can be produced on a very large scale by a mass production process, from microfabrication to chemical treatment, appear to be very promising analytical tools for proteomics research.
TiO2 Affinity Chromatography Microcolumn on Si Substrates for Phosphopeptide Analysis
Procedia Engineering, 2011
We present the fabrication and characterization of a TiO 2 affinity chromatography microcolumn on Si substrates to analyze phosphopeptides. The microfabricated column consisted of 32 parallel microchannels with common input and output and was fabricated by lithography and plasma etching of the Si substrate and sealed with lamination, while RF sputtering of TiO 2 target was used for producing the stationary phase. The chip was characterized by standard mono-phosphopeptide separation and detection with UV absorption spectroscopy. The chip design allows an expansion of its capacity by means of increasing the number of parallel microchannels at a constant sample volume.
International Journal of Mass Spectrometry, 2012
The applications of rapid screening benzodiazepines from urine and determining synthetic reaction products and kinetics semi-quantitatively using a microfabricated rotating multitip electrospray ionization (ESI) platform for mass spectrometry (MS) is presented. The ESI tips are based on lidless micropillar array ESI (PESI) sources where the transfer of liquid is based on capillary forces without external pumping. A single silicon platform contains 60 separate PESI tips which can be individually used by rotating the whole platform by six degrees each time from tip to tip using a computer control. The rotating multitip PESI platform with an ion trap mass spectrometer was successfully demonstrated in rapid identification and monitoring of intermediates and final products in chemical synthesis within 10 min. The system was also applied to high-throughput screening of benzodiazepines from urine samples. Urine samples were extracted with solid-phase extraction (SPE) using C 18 phase ZipTip TM pipettes, enabling the use as small sample volumes as 50 L of the urine sample. Therefore the whole sample treatment and the analysis with the rotating multitip PESI-MS took only 5 min per sample.