Extensive fractionation and identification of proteins within nasal lavage fluids from allergic rhinitis and asthmatic chronic rhinosinusitis patients

Allergic rhinitis (AR), chronic rhinosinusitis (CRS), and asthma are prevalent airway diseases that can have a substantial impact on a patient's quality of life. MS analyses of biological fluids can effectively screen for proteins associated with disease processes, however, initial detection of diagnostic proteins is difficult due to protein complexity and dynamic range. To enhance the detection of lower abundance proteins, intact nasal lavage fluid (NLF) proteins from nonpolypoid AR and from asthmatic CRS patients were extensively fractionated prior to LC/MS/MS analysis. Pooled NLF samples were processed to remove low molecular weight molecules and high abundance plasma proteins. Anion exchange (AX) chromatography followed by RP‐LC further separated the remaining intact NLF proteins. The resulting fractions were digested with trypsin and the peptides analyzed by LC/MS/MS. Spectra were searched with MASCOT, SEQUEST, and X!Tandem to obtain peptide identifications and subsequently analyzed by Scaffold software to identify parent proteins with at least 99% confidence. The 197 identified proteins are compared to those previously cited in the literature and the workflow evaluated to determine the usefulness for the detection of lower abundance proteins. This is the first extensive list of NLF proteins generated from CRS patients with coexisting asthma.     

Optimization of parameters for coverage of low molecular weight proteins

Proteins with molecular weights of <25 kDa are involved in major biological processes such as ribosome formation, stress adaption (e.g., temperature reduction) and cell cycle control. Despite their importance, the coverage of smaller proteins in standard proteome studies is rather sparse. Here we investigated biochemical and mass spectrometric parameters that influence coverage and validity of identification. The underrepresentation of low molecular weight (LMW) proteins may be attributed to the low numbers of proteolytic peptides formed by tryptic digestion as well as their tendency to be lost in protein separation and concentration/desalting procedures. In a systematic investigation of the LMW proteome of Escherichia coli, a total of 455 LMW proteins (27% of the 1672 listed in the SwissProt protein database) were identified, corresponding to a coverage of 62% of the known cytosolic LMW proteins. Of these proteins, 93 had not yet been functionally classified, and five had not previously been confirmed at the protein level. In this study, the influences of protein extraction (either urea or TFA), proteolytic digestion (solely, and the combined usage of trypsin and AspN as endoproteases) and protein separation (gel- or non-gel-based) were investigated. Compared to the standard procedure based solely on the use of urea lysis buffer, in-gel separation and tryptic digestion, the complementary use of TFA for extraction or endoprotease AspN for proteolysis permits the identification of an extra 72 (32%) and 51 proteins (23%), respectively. Regarding mass spectrometry analysis with an LTQ Orbitrap mass spectrometer, collision-induced fragmentation (CID and HCD) and electron transfer dissociation using the linear ion trap (IT) or the Orbitrap as the analyzer were compared. IT-CID was found to yield the best identification rate, whereas IT-ETD provided almost comparable results in terms of LMW proteome coverage. The high overlap between the proteins identified with IT-CID and IT-ETD allowed the validation of 75% of the identified proteins using this orthogonal fragmentation technique. Furthermore, a new approach to evaluating and improving the completeness of protein databases that utilizes the program RNAcode was introduced and examined.     

Hydride transfer from 1,4-dihydropyridines to pyridinium salts: Assessment of structural and energetic factors with hantzsch ester derived compounds

Hydride exchange occurs between 3,5 - di(alkoxycarbonyl) - 1,4 - dihydropyridines and their corresponding pyridinium salts. For the case of 1,2,6 - trimethyl - 3,5 - di(ethoxycarbonyl) - 1,4 - dihydropyridine in the presence of the structurally corresponding pyridinium perchlorate, hydride is transferred to the 4-position of the pyridinium salt in a reversible “blind” reaction as revealed by deuterium labeling experiments and to the 2,6-positions irreversibly to afford 1,2,6 - trimethyl - 3,5 - di(ethoxycarbonyl) - 1,2 - dihydropyridine as final product. Removal of the methyl groups at the 2,6-positions, i.e. 1 - methyl - 3,5 - di(methoxycarbonyl) - 1,4 -dihydropyridine and its structurally corresponding pyridium perchlorate, causes hydride transfer to become completely reversible. Substitution of the 4-position with Me, i.e. 1,2,4,6 - tetramethyl - 3,5 - di(methoxycarbonyl) -1,4- dihydropyridine and its corresponding pyridinium perchlorate leads to cessation of hydride transfer: the same is true for the analogous 4-phenyl (and substituted phenyl) compounds. However, these 1,4-dihydropyridines are capable of transferring hydride at reasonable temperatures to less highly substituted pyridinium salts. Activation parameters for some of these hydride transfers have been determined, mechanistic conclusions are presented, and the consequences of these observations for experiments with “model” NADH compounds are discussed.     

Laboratory simulations of the transformation of emmer wheat as a result of heating

Whole grains of emmer wheat were heated in a pre-heated tube oven at temperatures ranging from 130-700°C under controlled anoxic conditions for maximum 280 min. For each temperature a separate experiment was carried out. Physical properties including mass loss, thermal lag, external and internal morphology and the vitrinite reflectance, C and N content, and DTMS under CI (NH₃) and EI conditions were used to monitor changes as a function of the temperature. The results show remaining starch and protein rich material up to 250°C. From 310-400°C a secondary, thermally stable, product is formed and at higher temperatures a strongly carbon enriched tertiary product. This revised version was published online in August 2006 with corrections to the Cover Date.     

Normalization of relative peptide ratios derived from in-gel digests: applications to protein variant analysis at the peptide level

The ability to detect protein variants and post-translational modifications by mass spectrometry has become increasingly important. Unfortunately, the ability to detect variants in large intact proteins (>80,000 Da) is limited. Even in the analysis of smaller proteins, algorithms are required to determine the presence of a 2 Da mass shift in an intact 13 kDa protein because the isotopic distribution of the multiply charged ions of the variant overlaps the wild-type distribution. Fortunately, most modern instruments are capable of detecting variants in tryptic peptides derived from intact proteins. If a single common variant protein is known, the presence of a variant tryptic peptide can be easily demonstrated. A more difficult issue is the case where a multiplicity of peptides with multiple amino acid substitutions can be associated with pathology. In these cases a decrease in the relative amount of a variant peptide relative to other internal tryptic fragments would be diagnostic. However, the variability associated with the analysis of in-gel or solution digests of proteins, related to efficiencies in digestion, extraction and ionization, confounds variant analysis at the peptide level. A strategy was developed to normalize for this variability by utilizing multiple isotopically labeled internal standards for multiple peptides derived from the same protein. Erythrocyte spectrin from 36 normal and 25 abnormal osmotic fragility samples was analyzed as a test case. Three isotopically labeled target peptides comprising the alpha/beta-spectrin self-association sites were added to purified digested alpha-spectrin. The utilization of multiple internal standards demonstrates the capability to normalize for sample variability due to ionization efficiency, solvent effects, digestion and extraction efficiency.     

Perfluoroalkyl derivatives of chromium and cobalt containing sulphur donor ligands

Perfluoroalkyl derivates of chromium (III) have been prepared containing dithiocarbamate ligands, e.g. R_fCr(DTC)₂py. The complexes have a cis arrangement of pyridine and R_f groups as determined by the X-ray crystal structure determination of C₃F₇Cr(Me₂NCS₂)₂py. An analogous cobalt(III) complex C₃F₇Co(Et₂NCS₂)₂py has also been prepared in small yield and ¹⁹F NMR measurements indicate a similar structure.     

Vibrational interaction potentials

An expression is derived for the interaction potential arising from the correlative vibrational motion of fluctuating dipolar diatomic molecules, using as model the Morse oscillator. The theory is applied to two interacting NaCl molecules. The vibrational dipole-dipole coefficient,C ₆, is strongly temperature dependent with a value at room temperature of 3.788 × 10^?62 erg cm⁶.