Robust analysis of the yeast proteome under 50 kDa by molecular-mass-based fractionation and top-down mass spectrometry.

As the process of top-down mass spectrometry continues to mature, we benchmark the next installment of an improving methodology that incorporates a tube-gel electrophoresis (TGE) device to separate intact proteins by molecular mass. Top-down proteomics is accomplished in a robust fashion to yield the identification of hundreds of unique proteins, many of which correspond to multiple protein forms. The TGE platform separates 0-50 kDa proteins extracted from the yeast proteome into 12 fractions prior to automated nanocapillary LC-MS/MS in technical triplicate. The process may be completed in less than 72 h. From this study, 530 unique proteins and 1103 distinct protein species were identified and characterized, thus representing the highest coverage to date of the Saccharomyces cerevisiae proteome using top-down proteomics. The work signifies a significant step in the maturation of proteomics based on direct measurement and fragmentation of intact proteins.

Bilateral idiopathic sensorineural hearing loss following dental surgery.

BACKGROUND: This is a case study of an 18-year-old female who suffered a bilateral idiopathic sensorineural hearing loss that was coincident with the removal of four impacted wisdom teeth, Throughout childhood the patient had normal hearing for pure tones bilaterally as measured at the pediatrician's office. One month prior to dental surgery (May) the patient volunteered to participate in an auditory experiment at which time her pure-tone audiogram was normal. Immediately following surgery (June), the patient had substantial swelling of the face and complained of some hearing loss with no other auditory/vestibular complaints. The following month (July) during the course of a routine physical examination a pure-tone audiogram revealed bilateral, air-conduction thresholds of 30-35 dB HL (500-4000 Hz) and 20 dB HL (8000 Hz). Because bone conduction was not tested, it is impossible to know whether the hearing loss was conductive, mixed, or sensorineural. The pediatrician thought that the hearing loss was conductive and would resolve as the edema subsided, A month later (August) the subject again volunteered for an auditory experiment at which time her hearing again was tested. PURPOSE: The purpose of this report is to detail the dental procedures involved in the extraction of the wisdom teeth, to report the results of a variety and series of post-op hearing tests, and to discuss the possible mechanisms that might be involved in the "idiopathic" bilateral sensorineural hearing loss. RESEARCH DESIGN: Case report. RESULTS: During the August visit to the laboratory, hearing for pure tones bilaterally was 0 to 5 dB HL at 250-1000 Hz with a 40-45 dB HL notch at 2000 Hz with a return to 10 dB HL at 8000 Hz. Air conduction and bone conduction thresholds were equivalent. Word recognition in quiet was -92 percent correct for both ears, whereas the signal-to-noise ratio (SNR) hearing loss measured with the Words-in-Noise test was high normal in the left ear with a mild SNR hearing loss in the right ear. Tympanometry and acoustic reflex thresholds were normal. Distortion product otoacoustic emissions were reduced in the 1000-3000 Hz region for both ears, which is consistent with cochlear hearing loss. The hearing loss has remained unchanged for the past 19 months. CONCLUSIONS: The possible etiologies, including insults to the cochleae by vibration trauma and through alterations in the blood supply to the cochleae, are considered.

Parallel electrophoretic depletion, fractionation, concentration, and desalting of 96 complex biological samples for mass spectrometry.

The preparation of complex biological samples for high-throughput 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 high-throughput 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 reversed-phase 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.

Optically directed molecular transport and 3D isoelectric positioning of amphoteric biomolecules.

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.