The preparation of complex biological samples for highthroughput mass spectrometric analyses remains a significant bottleneck, limiting advancement of the capabilities of mass spectrometry (MS) and ultimately limiting development of novel clinical assays. The removal of interfering species (e.g., salts, detergents, and buffers), concentration of dilute analytes, and the reduction of sample complexity are required in order to maximize the quality of resultant MS data. This study describes a novel sample preparation method that makes use of electrophoresis to prepare complex biological samples for highthroughput MS analysis. The method provides for integration of key sample preparation steps, including depletion, fractionation, desalting, and concentration. The prepared samples are captured onto a monolithic reversedphase capture target that can be analyzed directly by a mass spectrometer. Up to 96 individual samples are simultaneously prepared for MS analysis in under 1 h. For standard proteins added to serum, this method provides femtomole level sensitivity and reproducible label-free detection (coefficient of variation <30%). This study demonstrates that this electrophoretic sample preparation system permits high-throughput sample preparation for mass spectrometric analysis of complex biological samples, such as serum, plasma, and tissue extracts.Mass spectrometry (MS) is fundamental to the analysis of a wide range of molecules due to its superb sensitivity and selectivity. 1-3 Soft ionization techniques such as electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI) now permit the accurate and precise measurement of the molecular mass of pharmaceuticals, metabolites, nucleic acids, and proteins, allowing for the identification, characterization, and quantitation of these biologically important analytes. Over the past decade, the capabilities of MS instrumentation dedicated to these applications has progressed significantly, now making it possible to analyze thousands of biological samples in a single day. 4 Despite the tremendous improvements in MS instrumentation, the presence of interfering chemical and biological materials compromise and can prevent MS analysis of specific analytes of interest. Taking advantage of the high-throughput capabilities of modern mass spectrometers is difficult without rapid and high-quality sample preparation techniques. 3,5 To fully exploit the analytical and economic advantages of modern mass spectrometry techniques in both biomedical research and clinical testing, high-throughput, high-fidelity, automated sample preparation methodologies are needed. 6 Biological samples that contain chemical contamination require a number of routine, often manual, sample preparation tasks prior to mass spectrometric analysis. The concentrations of salts, lipids, and nucleic acids in physiological samples as well as common laboratory reagents such as buffers and detergents contribute significantly to the overall molecular content of a biological sample and can ...
We demonstrate the formation of charged molecular packets and their transport within optically created electrical force-field traps in a pH-buffered electrolyte. We call this process photoelectrophoretic localization and transport (PELT). The electrolyte is in contact with a photoconductive semiconductor electrode and a counterelectrode that are connected through an external circuit. A light beam directed to coordinates on the photoconductive electrode surface produces a photocurrent within the circuit and electrolyte. Within the electrolyte, the photocurrent creates localized force-field traps centered at the illuminated coordinates. Charged molecules, including polypeptides and proteins, electrophoretically accumulate into the traps and subsequently can be transported in the electrolyte by moving the traps over the photoconductive electrode in response to movement of the light beam. The molecules in a single trap can be divided into aliquots, and the aliquots can be directed along multiple routes simultaneously by using multiple light beams. This photoelectrophoretic transport of charged molecules by PELT resembles the electrostatic transport of electrons within force-field wells of solid-state charge-coupled devices. The molecules, however, travel in a liquid electrolyte rather than a solid. Furthermore, we have used PELT to position amphoteric biomolecules in three dimensions. A 3D pH gradient was created in an electrolyte medium by controlling the illumination position on a photoconductive anode where protons were generated electrolytically. Photoelectrophoretic transport of amphoteric molecules through the pH gradient resulted in accumulation of the molecules at their apparent 3D isoelectric coordinates in the medium. (1) first used a single light beam to create a force gradient that trapped suspended dielectric particles. This optical trapping technique works optimally for Ϸ1-m particles (2). Such devices, commonly called optical tweezers, are quite versatile and additionally have been used to build 3D nanostructures and to control the spinning of trapped particles with optically polarized light (3, 4). In practice, however, optical tweezers cannot transport objects smaller than the wavelength of light. This includes many biomolecules e.g., DNA fragments, oligonucleotides, proteins, and peptides. Instead, such small molecules must first be attached to larger particles called ''handles'' (5).¶ Another technique, called the ''optoelectronic tweezers,'' has been used to position single particles with intensity-modulated light directed onto a semiconductor electrode (6). This alternative optoelectronic technique utilizes dielectrophoresis wherein the modulated electric field induces transient electrical dipoles in the particles. Both methods, however, are limited to transporting large particles.We demonstrate light-driven transport of proteins without attachment to larger particles. We use a beam of light directed onto a photoconductive semiconductor surface in contact with an electrolyte medium to create con...
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