An online nonenzymatic digestion method utilizing a microwave-heated flow cell and mild acid hydrolysis at aspartic acid (D) for rapid protein identification is described. This methodology, here termed microwave D-cleavage, was tested with proteins ranging in size from 5 kDa (insulin) to 67 kDa (bovine serum albumin) and a bacterial cell lysate ( Escherichia coli). A microwave flow cell consisting of a 5 microL total volume reaction loop connected to a sealed reaction vessel was introduced into a research grade microwave oven. With this dynamic arrangement, the injected sample was subjected to microwave radiation as it flowed through the reaction loop and was digested in less than 5 min. Different digestion times can be achieved by varying the sample flow rate and/or length of the loop inside the microwave flow cell. The microwave flow cell can be operated individually with the output being collected for matrix assisted laser ionization/desorption (MALDI) mass spectrometry (MS) or connected online for liquid chromatography (LC) electrospray ionization (ESI)-MS. In the latter configuration, the microwave flow cell eluates containing digestion products were transferred online to a reversed phase liquid chromatography column for direct ESI-MS and ESI-MS/MS analyses (specifically, Collision Induced Dissociation, CID). Concurrently with the microwave D-cleavage step, disulfide bond reduction/cleavage was achieved by the coinjection of dithiothreitol (DTT) with the sample prior to online microwave heating and online LC-MS analysis and so eliminating the need for alkylation of the reduced protein. All protein standards, protein mixtures, and proteins in a bacterial cell lysate analyzed by this new online methodology were successfully identified via a SEQUEST database search of fragment ion mass spectra. Overall, online protein digestion and identification was achieved in less than 40 min total analysis time, including the chromatographic step.

The nonenzymatic digestion of proteins by microwave D-cleavage is an effective technique for site-specific cleavage at aspartic acid (D). This specific cleavage C-terminal to D residues leads to inherently large peptides (15-25 amino acids) that are usually relatively highly charged (above +3) when ionized by electrospray ionization (ESI) due to the presence of several basic amino acids within their sequences. It is well-documented that highly charged peptide ions generated by ESI are well-suited for electron transfer dissociation (ETD), which produces c- and z-type fragment ions via gas-phase ion/ion reactions. In this paper, we describe the sequence analysis by ETD tandem mass spectrometry (MS/MS) of multiply charged peptides generated by microwave D-cleavage of several standard proteins. Results from ETD measurements are directly compared to CID MS/MS of the same multiply charged precursor ions. Our results demonstrate that the nonenzymatic microwave D-cleavage technique is a rapid (<6 min) and specific alternative to enzymatic cleavage with Lys-C or Asp-N to produce highly charged peptides that are amenable to informative ETD.

We report an online non-enzymatic method for site-specific digestion of proteins to yield peptides that are well suited for collision induced dissociation (CID) tandem mass spectrometry (MS/MS). The method combines online microwave heating acid hydrolysis at aspartic acid and online electrochemical oxidation at tryptophan and tyrosine. The combined microwave/electrochemical (microwave/echem) digestion is reproducible and produces peptides with an average sequence length of 10 amino acids. This peptide length is similar to the average peptide length of 9 amino acids obtained by digestion of proteins with the enzyme trypsin. As a result, the peptides produced by this novel non-enzymatic digestion method, when analyzed by ESI-MS, produce protonated molecules with mostly +1 and +2 charge states. The combination of these two non-enzymatic methods overcomes shortcomings with each individual method in that: i) peptides generated by the microwave-hydrolysis method have an average amino acid length of 16 amino acids, and ii) the inability of the electrochemical-cleavage method to reproducibly digest proteins with molecular masses above 4 kDa. Preliminary results are presented on the application and utility of this rapid online digestion (total of 6 min digestion time) on a series of standard peptides and proteins as well as an E. coli protein extract.

Czochralski-grown (cz) silicon samples have been implanted at room temperature with low doses (109-10" cm-') of "B and "Si using energies between 0.36 and 3.0 MeV. Deep-level transient spectroscopy has been applied for sample analysis, and four levels -0.18, -0.23, -0.35 and -0.43 eV below the conduction band edge (E,) are resolved. The concentrations of these levels have been determined as a function of ion dose and sample depth. The total concentration of electrically active defects amounts to less than 10% of the vacancy concentration predicted by Monte Carlo simulations. The depth profiles of the levels at E, -0.18, E, -0.23 and E. -0.43 eV are mainly confined to the damage peak region, and evidence is obtained for substantial surface-enhanced annihilation of migrating monovacancies. However, the depth distribution of the E, -0.35 eV level exhibits a pronounced leading surface tall and is broader than expected from energy deposition calculations. The identity of this latter level is not well established but we argue that the generation process involves diffusion of interstitial carbon. Finally, in contrast to that for cz samples irradiated with MeV electrons t h e strengths of the E, -0.23 and E, -0.43 eV levels deviate from a one-to-one proportionality. Similar deviations have previously been observed in ion-implanted float-zone samples and are attributed to strain accommodated by the lattice in t h e damage peak region.

The thermally-induced Co/SixGe1-x reaction has been studied for a series of isochronal (25–600°C/20 min) and isothermal (600°C/u-240 min) annealing sequences using Rutherford backscattering spectrometry, transmission electron microscopy and sheet resistance measurements. Annealing at 600°C yields a reacted surface layer comprised of Si-rich CoSixGe1-x, Ge-rich SiyGe1-y and possibly CoSi2, with the two former constituents exhibiting a degree of epitaxial alignment with the substrate. The formation of Co/SiSixGe1-x alloys is discussed in terms of the ternary phase diagram.