Fully automated isotopic dimethyl labeling and phosphopeptide enrichment using a microfluidic HPLC phosphochip.

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

Exploring the human leukocyte phosphoproteome using a microfluidic reversed-phase-TiO2-reversed-phase high-performance liquid chromatography phosphochip coupled to a quadrupole time-of-flight mass spectrometer.

The study of protein phosphorylation events is one of the most important challenges in proteome analysis. Despite the importance of phosphorylation for many regulatory processes in cells and many years of phosphoprotein and phosphopeptide research, the identification and characterization of phosphorylation by mass spectrometry is still a challenging task. Recently, we introduced an approach that facilitates the analysis of phosphopeptides by performing automated, online, TiO(2) enrichment of phosphopeptides prior to mass spectrometry (MS) analysis. The implementation of that method on a "plug-and-play" microfluidic high-performance liquid chromatography (HPLC) chip design will potentially open up efficient phosphopeptide enrichment methods enabling phosphoproteomics analyses by a broader research community. Following our initial proof of principle, whereby the device was coupled to an ion trap, we now show that this so-called phosphochip is capable of the enrichment of large numbers of phosphopeptides from complex cellular lysates, which can be more readily identified when coupled to a higher resolution quadrupole time-of-flight (Q-TOF) mass spectrometer. We use the phosphochip-Q-TOF setup to explore the phosphoproteome of nonstimulated primary human leukocytes where we identify 1012 unique phosphopeptides corresponding to 960 different phosphorylation sites providing for the first time an overview of the phosphoproteome of these important circulating white blood cells

Fully automated isotopic dimethyl labeling and phosphopeptide enrichment using a microfluidic HPLC phosphochip.

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

Chip-Based Enrichment and NanoLC-MS/MS Analysis of Phosphopeptides from Whole Lysates.

Protein phosphorylation may be the most widespread and possibly most important post-translational modification (PTM). Considering such a claim, it should be no surprise that huge efforts have been made to improve methods to allow comprehensive study of cellular phosphorylation events. Nevertheless, comprehensive identification of sites of protein phosphorylation is still a challenge, best left to experienced proteomics experts. Recent advances in HPLC chip manufacturing have created an environment to allow automation of popular techniques in the bioanalytical world. One such tool that would benefit from the increased ease and confidence brought by automated 'nanoflow' analysis is phosphopeptide enrichment. To this end, we have developed a reusable HPLC nanoflow rate chip using TiO 2 particles for selective phosphopeptide enrichment. Such a design proved robust, easy to use, and was capable of consistent performance over tens of analyses including minute amounts of complex cellular lysates

Chip-Based Enrichment and NanoLC-MS/MS Analysis of Phosphopeptides from Whole Lysates.

Protein phosphorylation may be the most widespread and possibly most important post-translational modification (PTM). Considering such a claim, it should be no surprise that huge efforts have been made to improve methods to allow comprehensive study of cellular phosphorylation events. Nevertheless, comprehensive identification of sites of protein phosphorylation is still a challenge, best left to experienced proteomics experts. Recent advances in HPLC chip manufacturing have created an environment to allow automation of popular techniques in the bioanalytical world. One such tool that would benefit from the increased ease and confidence brought by automated 'nanoflow' analysis is phosphopeptide enrichment. To this end, we have developed a reusable HPLC nanoflow rate chip using TiO 2 particles for selective phosphopeptide enrichment. Such a design proved robust, easy to use, and was capable of consistent performance over tens of analyses including minute amounts of complex cellular lysates

Flow-Cell-Induced Dispersion in Flow-through Absorbance Detection Systems: True Column Effluent Peak Variance

Following a brief overview of the emergence of absorbance detection in liquid chromatography, we focus on the dispersion caused by the absorbance measurement cell and its inlet. A simple experiment is proposed wherein chromatographic flow and conditions are held constant but a variable portion of the column effluent is directed into the detector. The temporal peak variance (σ_t,obs²), which increases as the flow rate (<i>F</i>) through the detector decreases, is found to be well-described as a quadratic function of ¹/_<i>F</i>. This allows the extrapolation of the results to zero residence time in the detector and thence the determination of the true variance of the peak prior to the detector (this includes contribution of all preceding components). This general approach should be equally applicable to detection systems other than absorbance. We also experiment where the inlet/outlet system remains the same but the path length is varied. This allows one to assess the individual contributions of the cell itself and the inlet/outlet system.to the total observed peak. The dispersion in the cell itself has often been modeled as a flow-independent parameter, dependent only on the cell volume. Except for very long path/large volume cells, this paradigm is simply incorrect

Width Based Quantitation of Chromatographic Peaks: Principles and Principal Characteristics

Height- and area-based quantitation reduce two-dimensional data to a single value. For a calibration set, there is a single height- or area-based quantitation equation. High-speed high-resolution data acquisition now permits rapid measurement of the width of a peak (<i>W</i>_<i>h</i>), at any height <i>h</i> (a fixed height, not a fixed fraction of the peak maximum) leading to any number of calibration curves. We propose a width-based quantitation (WBQ) paradigm complementing height or area based approaches. When the analyte response across the measurement range is not strictly linear, WBQ can offer superior overall performance (lower root-mean-square relative error over the entire range) compared to area- or height-based linear regression methods, rivaling weighted linear regression, provided that response is uniform near the height used for width measurement. To express concentration as an explicit function of width, chromatographic peaks are modeled as two different independent generalized Gaussian distribution functions, representing, respectively, the leading/trailing halves of the peak. The simple generalized equation can be expressed as <i>W</i>_<i>h</i> = <i>p</i>(ln <i>h̅</i>)^<i>q</i>, where <i>h̅</i> is <i>h</i>_max/<i>h</i>, <i>h</i>_max being the peak amplitude, and <i>p</i> and <i>q</i> being constants. This fits actual chromatographic peaks well, allowing explicit expressions for <i>W</i>_<i>h</i>. We consider the optimum height for quantitation. The width-concentration relationship is given as ln <i>C</i> = <i>aW</i>_<i>h</i>^<i>n</i> + <i>b</i>, where <i>a</i>, <i>b</i>, and <i>n</i> are constants. WBQ ultimately performs quantitation by projecting <i>h</i>_max from the width, provided that width is measured at a fixed height in the linear response domain. A companion paper discusses several other utilitarian attributes of width measurement

Micromachined Fused Silica Liquid Core Waveguide Capillary Flow Cell

A planar, chip-based flow cell for UV–vis absorbance detection in HPLC is presented. The device features a microfabricated free-standing liquid core waveguide (LCW) capillary detection tube of long path length that is based on total internal reflection. We report on the linearity and calibration slope characteristics of lithographically produced LCWs with different interior/exterior geometries. 3D ray tracing was indispensable in modeling behavior in the more demanding geometries: multipath behavior may be intrinsic to these waveguides with consequent nonlinearity. Fortunately, nonlinearity in lithographically easy-to-produce waveguide geometries (such as with a flat, concave exterior and a round interior) is not as detrimental as might be initially expected. Experimental performance is predictably affected by the attainable surface quality of the LCW and efficient and reproducible coupling of the input light into the LCW