Highly complex protein mixtures can be directly analyzed after proteolysis by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). In this paper, we have utilized the combination of strong cation exchange (SCX) and reversed-phase (RP) chromatography to achieve two-dimensional separation prior to MS/MS. One milligram of whole yeast protein was proteolyzed and separated by SCX chromatography (2.1 mm i.d.) with fraction collection every minute during an 80-min elution. Eighty fractions were reduced in volume and then re-injected via an autosampler in an automated fashion using a vented-column (100 µm i.d.) approach for RP-LC-MS/MS analysis. More than 162 000 MS/MS spectra were collected with 26 815 matched to yeast peptides (7537 unique peptides). A total of 1504 yeast proteins were unambiguously identified in this single analysis. We present a comparison of this experiment with a previously published yeast proteome analysis by Yates and colleagues (Washburn, M. P.; Wolters, D.; Yates, J. R., III. Nat. Biotechnol. 2001, 19, 242-7). In addition, we report an in-depth analysis of the false-positive rates associated with peptide identification using the Sequest algorithm and a reversed yeast protein database. New criteria are proposed to decrease false-positives to less than 1% and to greatly reduce the need for manual interpretation while permitting more proteins to be identified.
A micromachining process is described for fabricating a mass spectrometry electrospray source on a silicon chip. The process utilizes polymer (parylene) layers to form a system of chambers, filters, channels, and hollow needle structures (electrospray emitters) that extend more than a millimeter beyond the edge of the silicon substrate. The use of photoresist as the sacrificial layer facilitates the creation of long channels. Access to the channel structures on the chip is through a port etched through the silicon substrate that also serves as a sample reservoir. A reusable chip holder consisting of two plastic plates and an elastomer gasket provides the means to mount the chip in front of the mass spectrometer inlet and make electrical and gas connections. The electrospray emitters have tapered tips with 5 microns x 10 microns rectangular openings. The shape of the tip can be varied depending on the shape of the mask used to protect the parylene structures during the final plasma etch. The parylene emitters are physically robust and require only a high electric field to achieve stable electrospray operation over a period of a few hours. Direct comparisons with conventional glass or fused silica emitters indicated very similar performance with respect to signal strength and stability, spectral quality, and endurance. The automated MS/MS analysis of a mixture of tryptic peptides was no more difficult and yielded nearly identical results as the analysis of the same sample using a conventional nanospray device. This work demonstrates that an efficient electrospray interface to mass spectrometry can be integrated with other on-chip structures and mass-produced using a batch process.
A methodology is described for creating a monolithic chromatography support within a pulled fused-silica electrospray needle. The monolith was formed from a mixture of styrene, divinylbenzene, 1-dodecanol, and toluene using 2,2'-azobis(isobutyronitrile) as the catalyst. The mixture was loaded into 150-micron-i.d. fused-silica capillary tubing with a pulled 5-10-micron needle tip at one end. Polymerization at 65 degrees C followed by removal of the porogen material yielded a stable, porous, monolithic support which had excellent properties for the separation and on-line, electrospray, mass spectrometry analysis of peptides and proteins. The performance of the monolith-filled electrospray needles was compared with similar needles filled with commercial C18 silica and polymeric particulate supports. Separation efficiencies for both protein and peptide mixtures were generally equal to or better than the particulate supports at comparable pressures and flow rates. The ion chromatograms derived from the on-line MS analysis were remarkably free from chemical background signals that often complicate the LC/MS analysis of femtomole amounts of sample. Good sequence coverage was obtained by LC/MS/MS analysis of the peptide mixture obtained from a protein isolated by silver-stained gel electrophoresis. The capability of the monolith to do peak parking experiments was demonstrated by the characterization of an immunoreactive HPLC fraction. The simple fabrication method, chromatographic performance, and robust nature of these microscale integrated column electrospray sources make them ideally suited for high-sensitivity tandem LC/MS analyses.
We have designed a simple system to allow visualization of electroosmotlc flow across a capillary junction. Two capillaries are placed In close proximity by careful micropositioning, and the distance between the capillaries Is controlled with submicron resolution. The Junction thus produced Is placed at the focal point of a stereomicroscope, where the Image collected by the microscope Is Imaged onto a CCD camera. Illumination of the Junction (Immersed In a buffer solution) allows visualization of the electroosmosis of a dye across the liquid Junction. This has allowed the determination of the dimensional characteristics of the Junction and the optimization of the composition of the buffer bathing the Junction, thereby allowing efficient transfer of a sample across a 50-200-jum gap.
Application of mild vibration to an immobilized trypsin capillary microreactor can enhance digestion rates for many globular and glycosylated proteins (12-70-kDa range) without additional sample handling. A sinusoid wave form generator and a simple piezoelectric transducer were used to apply vibration in a wide frequency range to the 50-μm-i.d. enzyme microreactor over its entire length. The mass transport properties of the microreactor were quantitatively examined for protein digestions through the use of an artificial globular protein. This was synthesized by covering the surface of 35-nm-diameter latex beads with a peptide (Leu-Arg-Leu). Capillary electrophoresis analysis of the microreactor products showed there were no mass transport-related effects for vibration of the capillary. Digestions of a range of globular protein structures were performed and the products analyzed by capillary electrophoresis. The rate enhancements were found to be related to the stability of the protein tertiary and secondary structure. Cytochrome c showed a dramatic acceleration in rate of digestion as the vibration frequency increased over a range of 200 Hz to 7.1 kHz. The ability to enhance reaction rates for very stable proteins can be gained by additional means of destabilizing the protein, as shown by removal of calcium from α-lactalbumin. Vibration of the enzyme capillary will have the greatest utility for extremely limited protein samples since chemical modification to completely denature proteins usually requires considerable sample handling.
AILERON is developing a selective approach to restore programmed cell death in cancer cells by borrowing from nature the BH3 domain alpha-helical “keys” from pro-apoptotic members of the BCL-2 family that fit into the “locks” of other anti- and pro-apoptotic BCL-2 proteins. AILERON has applied its proprietary chemical strategy termed hydrocarbon stapling (J. Am. Chem. Soc., 2000 122:5891) to generate cell-permeable BH3 stapled peptides that modulate the intracellular protein-protein interactions of BCL-2 family members and selectively kill cancer cells both in vitro and in vivo (Science, 2004 305:1466). The lead compounds possess the innate characteristics of the endogenous peptides they mimic, including mechanism of action and target specificity. BID BH3 and BIM BH3 stapled peptides have recently been shown to directly activate the pro-death molecule BAX, a unique property that differentiates BH3 stapled peptides from all BCL-2 small molecule antagonists (Mol. Cell, 2006 24:199). BID BH3 and BIM BH3 stapled peptides were tested for their ability to induce programmed cell death in a panel of 12 lymphoid-derived tumor cell lines. T-ALL derived cell lines were the most sensitive to the compounds, followed by multiple myeloma lines. CML-derived cell lines were the least sensitive. Resting human peripheral blood lymphocytes (hPBLs) and normal human embryonic lung fibroblasts (WI-38) were found to be resistant to BH3 stapled peptides, indicating that the compound may target only cells that are “primed to die” and not normal “resting” cells. The anti-cancer activity of BH3 stapled peptides was further investigated in orthotopic xenograft models and shown to dramatically suppress tumor growth. In a mixed lineage leukemia model (SEMK2), a tumor over control (T/C) of 27% was observed after 13 days of treatment at 30 mg/Kg IV q.d. We also investigated whether BH3 stapled peptides elicit an antibody response in rodents. No antibody titer was detected, indicating that BH3 stapled peptides are non-antigenic in rodents. While peptides are oftentimes unstable in vivo, with half-lives typically in the range of a few minutes, BH3 stapled peptides were 100% stable in both mouse and human plasma ex vivo, and exhibited excellent PK profiles in rats with half-lives greater than three hours. BH3 stapled peptides were well tolerated in all animal models tested to date. In conclusion, we show that BH3 stapled peptides exhibit promising pharmacological properties and represent a novel class of drugs for the treatment of hematological malignancies.
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