Peptide extraction from formalin-fixed paraffin-embedded tissue.

This unit describes how to extract tryptic peptides from formalin-fixed, paraffin-embedded (FFPE) tissues for analysis using nano-reverse-phase liquid chromatography/tandem mass spectrometry (nRPLC-MS/MS). The tissues are deparaffinized in the first protocol. Following deparaffinization, the cells are harvested via one of two methods: needle dissection or laser capture microdissection (LCM). Needle dissection is performed using hydrated, unstained tissue, whereas LCM is performed with dehydrated, hematoxylin-stained tissue. Heat is applied to the collected cells to reverse the cross-links that have formed during the formalin fixation process. Finally, the cells are digested using filter-aided sample preparation, in which buffers are exchanged throughout the process. An alternate protocol using commercially available Liquid Tissue is also described. These samples are then ready for mass spectrometric analysis.

Composition domains in monoterpene secondary organic aerosol.

The composition and structure of freshly formed oligomers in alpha- and beta- pinene SOA are studied with high performance mass spectrometry to provide insight into the SOA formation mechanism. Van Krevelen plots (H:C ratio vs O:C ratio) are interpreted in the context of distinct structural domains that correspond to separate oligomer formation routes. The domain containing most of the signal intensity encompasses elemental formulas that correspond to oligomerization reactions of intermediates and/or stable molecule monomers produced by ozonolysis of the precursor. While oligomers involving reactive intermediates from the hydroperoxide channel dominate the product distribution, products are also observed that uniquely map to the stable Criegee intermediate and/ or combinations of stable molecule monomers. A second domain encompasses molecules having lower H:C ratios but similar O:C ratios to the first domain. Many of the products observed in this domain have double bond equivalents greater than the maximum number possible when forming dimers by standard reaction mechanisms and are interpreted in the context of repeated self-reactions of alkoxy/peroxy radicals. A third domain encompasses molecules having very high H:C and O:C ratios consistent with polymerization of formaldehyde and/or acetaldehyde. These domains remain distinguishable from experiment to experiment and among different extraction solvents (50/50 methanol-water, 50/50 acetonitrile-water,100% water).

Reactive uptake of trimethylamine into ammonium nitrate particles.

The reaction of trimethylamine (TMA) vapor with a polydisperse distribution of ammonium nitrate particles (20-500 nm dia.) was studied in a flow tube reactor with particle analysis by laser desorption (1064 nm) 70 eV electron ionization (EI) in an ion trap time-of-flight (IT-TOF) aerosol mass spectrometer. When the TMA vapor concentration was very high, essentially complete exchange of TMA for ammonia in the particles was achieved. When the TMA vapor concentration was lower ( approximately 500 ppb for a reaction time of 23 s), partial exchange was observed, and the initial reactive uptake coefficient was estimated to be on the order of 2 x 10(-3) at 20% RH. This value suggests that measurable exchange is possible in the atmosphere when particles are exposed to an amine concentration on the order of 1 ppb for a few hours. The effects of particle size, water content, and amine molecular structure on uptake remain to be elucidated.

Oligomers in the early stage of biogenic secondary organic aerosol formation and growth.

The formation of secondary organic aerosol (SOA) by reaction of ozone with monoterpenes (beta-pinene, delta3-carene, limonene, and sabinene) was studied on a short time scale of 3-22 s with a flow tube reactor. Online chemical analysis was performed with the Photoionization Aerosol Mass Spectrometer (PIAMS) to obtain molecular composition and the Nanoaerosol Mass Spectrometer (NAMS) to obtain elemental composition. Molecular composition data showed that dimers and higher order oligomers are formed within seconds after the onset of reaction, indicating that there is no intrinsic kinetic barrier to oligomer formation. Because oligomer formation is fast, it is unlikely that a large number of steps are involved in their formation. Therefore, ion distributions in the PIAMS spectra were interpreted through reactions of intermediates postulated in previous studies with monomer end products or other intermediates. Based on ion signal intensities in the mass spectra, organic peroxides appear to comprise a greater fraction of the aerosol than secondary ozonides. This conclusion is supported by elemental composition data from NAMS that gave C:O ratios in the 2.2-2.7 range.

Chemistry of particle inception and growth during alpha-pinene ozonolysis.

A flow-tube reactor was used to study the formation of particles from alpha-pinene ozonation. Particle phase products formed within the first 3-22 s of reaction were analyzed online using a scanning mobility particle sizer and two particle mass spectrometers. The first, a photoionization aerosol mass spectrometer (PIAMS), was used to determine the molecular composition of nascent particles between 30 and 50 nm in diameter. The second, a nano-aerosol mass spectrometer (NAMS), was used to determine the elemental composition of individual particles from 50 nm to below 10 nm in diameter. Molecular composition measurements with PIAMS confirm that both the stabilized Criegee intermediate and hydroperoxide channels of alpha-pinene ozonolysis are operative. However, these channels alone cannot explain the high oxygen content of the particles measured with NAMS. The carbon-to-oxygen mole ratios of suspected nucleating agents are in the range of 2.25-4.0, while the measured ratios are from 1.9 for 9 nm particles to 2.5 and 2.7 for 30 and 50 nm particles, respectively. The large oxygen content may arise by cocondensation of small oxygenated molecules such as water or multistep reactions with ozone, water, or other species that produce highly oxygenated macromolecules. In either case, the increasing ratio with increasing particle size suggests that the aerosol becomes less polar with time.