The proteome of normal male urine from a commercial pooled source has been examined using direct liquid chromatography-tandem mass spectrometry (LC-MS/MS). The entire urinary protein mixture was denatured, reduced and enzymatically digested prior to LC-MS/MS analysis using a hybrid-quadrupole time-of-flight mass spectrometer (Q-TOF) to perform data-dependent ion selection and fragmentation. To fragment as many peptides as possible, the mixture was analyzed four separate times, with the mass spectrometer selecting ions for fragmentation from a subset of the entire mass range for each run. This approach requires only an autosampler on the HPLC for automation (i.e, unattended operation). Across these four analyses, 1.450 peptide MS/MS spectra were matched to 751 sequences to identify 124 gene products (proteins and translations of expressed sequence tags). Interestingly, the experimental time for these analyses was less than that required to run a single two-dimensional gel.
The agouti-related protein gene (Agrp) plays an important role in body weight regulation. The mature human protein is a single polypeptide chain of 112 amino acid residues, consisting of an N-terminal acidic region and a unique C-terminal cysteine-rich domain. The disulfide structure of recombinant human AGRP was determined by chemical methods using partial reduction with tris(2-carboxyethyl)phosphine under acidic conditions, followed by direct alkylation with N-ethylmaleimide or fluorescein-5-maleimide. Partial reduction and alkylation provided several forms of AGRP that were modified in a stepwise fashion. The resulting proteins were characterized by peptide mapping, sequence analysis, and mass spectrometry, showing that AGRP contained a highly reducible disulfide bond, C85-C109, followed by less reactive ones, C90-C97, C74-C88, C67-C82, and C81-C99, respectively. The chemically defined disulfide connectivity of the recombinant human AGRP was homologous to that of omega-agatoxin IVB except for an additional disulfide bond, C85-C109.
Highly concentrated human recombinant interleukin-1 receptor antagonist (IL-1ra) aggregates at elevated temperature without perturbation in its secondary structure. The protein aggregation can be suppressed depending on the buffer ionic strength and the type of anion present in the sample solution. Phosphate is an approximately 4-fold weaker suppressant than either citrate or pyrophosphate on the basis of the measured protein aggregation rates. This is in agreement with the strength of protein-anion interactions at the IL-1ra single anion-binding site as judged by the estimated dissociation constant values of 2.9 mM, 3.8 mM, and 13.7 mM for pyrophosphate, citrate, and phosphate, respectively. The strength of binding also correlates with the anion size and with the number of ionized groups available per molecule at a given pH. Affinity probing of IL-1ra with methyl acetyl phosphate (MAP) in combination with proteolytic digestion and mass spectral analysis show that an anion-binding site location on the IL-1ra surface is contributed by lysine-93 and lysine-96 of the loop 84-98 as well as by lysine-6 of the unstructured N-terminal region 1-7. The replacement of lysine-93 with alanine by site-directed mutagenesis results in dramatically suppressed IL-1ra aggregation. Furthermore, when the unstructured N-terminal region of IL-1ra is removed by limited proteolysis, a 2-fold increase in the time course of the aggregation lag phase is observed for the truncated protein. An anion-controlled mechanism of IL-1ra aggregation is proposed by which the anion competition for the protein cationic site prevents formation of intermolecular cation-pi interactions and, thus, interferes with the protein asymmetric self-association pathway.
With an emphasis on obtaining a multitude of high quality tandem mass spectrometry spectra for protein identification, instrumental parameters are described for the liquid chromatography-tandem mass spectrometry analysis of trypsin digested unfractionated urine using a hybrid quadrupole-time-of-flight (Q-TOF) mass spectrometer. Precursor acquisition rates of up to 20 distinct precursors/minute in a single analysis were obtained through the use of parallel precursor selection (four precursors/survey period) and variable collision induced dissociation integration time (1 to 6 periods summed). Maximal exploitation of the gas phase fractionated ions was obtained through the use of narrow survey scans and iterative data-dependent analyses incorporating dynamic exclusion. The impact on data fidelity as a product of data-dependent selection of precursor ions from a dynamically excluded field is discussed with regards to sample complexity, precursor selection rates, survey scan range and facile chemical modifications. Operational and post-analysis strategies are presented to restore data confidence and reconcile the greatest number of matched spectra.
and Moody, 1976). Also, when legumes are used as a living mulch, they can supply N to the main crop (Scott Previous research has shown that with adequate suppression, kura et al., 1987). clover (Trifolium ambiguum M. Bieb.) can be managed as a living mulch in corn (Zea mays L.); however, significant yield loss was Corn production in living mulch and interseeded sysobserved in some environments. This study evaluated two herbicide-tems typically results in yield loss in the North-Central resistant corn hybrids at three levels of kura clover living mulch USA from competition for moisture and N (Kurtz et suppression over multiple environments. In 1999 and 2000 near Arlingal., 1952; Pendleton et al., 1957). Fisher and Burrill ton and in 2000 near Lancaster, WI, glyphosate [N-(phosphonometh-(1993) and Zemenchik et al. (2000) also note that cool yl)glycine]-resistant corn (Roundup Ready corn, RRC) and glufosispring temperatures could reduce corn yields because nate [2-amino-4-(hydroxymethylphosphinyl) butanoic acid]-resistant of delayed planting or by giving a cool-season clover a corn (Liberty Link corn, LLC) hybrids were planted where kura clover competitive advantage over corn, a warm-season grass. had been (i) killed for monocrop corn, (ii) strongly suppressed with The key to the successful use of living mulches for glyphosate and dicamba (3,6-dichloro-2-methoxybenzoic acid), or (iii) corn production is controlling competition from the lightly suppressed with only glyphosate. Suppressed kura clover also had a 25-cm clopyralid (3,6-dichloro-2-pyridinecarboxylic acid) plus mulch crop. However, if the mulch suppression is excesdicamba-killed band into which corn was planted. Subsequent post-sive, it will not recover. Zemenchik et al. (2000) showed emergence applications of glyphosate or glufosinate herbicide were that corn could be grown in a kura clover living mulch made for each hybrid. Corn whole-plant yield ranged from 17.3 to when adequately suppressed by herbicide, without re-19.9 Mg ha Ϫ1 , and grain yield ranged from 10.8 to 12.3 Mg ha Ϫ1 .duced corn whole-plant or grain yields. In this system, Yield of whole-plant and grain across both corn hybrids did not differ kura clover will recover to full production within 12 mo between monocrop corn and corn in strongly suppressed kura clover. of corn harvest. However, corn performance in the living Whole-plant yield of monocrop corn was 8 to 11% greater and grain
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