The SOS response aids bacterial propagation by inhibiting cell division during repair of DNA damage. We report that inactivation of the ftsI gene product, penicillin binding protein 3, by either beta-lactam antibiotics or genetic mutation induces SOS in Escherichia coli through the DpiBA two-component signal transduction system. This event, which requires the SOS-promoting recA and lexA genes as well as dpiA, transiently halts bacterial cell division, enabling survival to otherwise lethal antibiotic exposure. Our findings reveal defective cell wall synthesis as an unexpected initiator of the bacterial SOS response, indicate that beta-lactam antibiotics are extracellular stimuli of this response, and demonstrate a novel mechanism for mitigation of antimicrobial lethality.

We carried out a test sample study to try to identify errors leading to irreproducibility, including incompleteness of peptide sampling, in LC-MS-based proteomics. We distributed a test sample consisting of an equimolar mix of 20 highly purified recombinant human proteins, to 27 laboratories for identification. Each protein contained one or more unique tryptic peptides of 1250 Da to also test for ion selection and sampling in the mass spectrometer. Of the 27 labs, initially only 7 labs reported all 20 proteins correctly, and only 1 lab reported all the tryptic peptides of 1250 Da. Nevertheless, a subsequent centralized analysis of the raw data revealed that all 20 proteins and most of the 1250 Da peptides had in fact been detected by all 27 labs. The centralized analysis allowed us to determine sources of problems encountered in the study, which include missed identifications (false negatives), environmental contamination, database matching, and curation of protein identifications. Improved search engines and databases are likely to increase the fidelity of mass spectrometry-based proteomics.

MS/MS and associated database search algorithms are essential proteomic tools for identifying peptides. Due to their widespread use, it is now time to perform a systematic analysis of the various algorithms currently in use. Using blood specimens used in the HUPO Plasma Proteome Project, we have evaluated five search algorithms with respect to their sensitivity and specificity, and have also accurately benchmarked them based on specified false-positive (FP) rates. Spectrum Mill and SEQUEST performed well in terms of sensitivity, but were inferior to MASCOT, X!Tandem, and Sonar in terms of specificity. Overall, MASCOT, a probabilistic search algorithm, correctly identified most peptides based on a specified FP rate. The rescoring algorithm, PeptideProphet, enhanced the overall performance of the SEQUEST algorithm, as well as provided predictable FP error rates. Ideally, score thresholds should be calculated for each peptide spectrum or minimally, derived from a reversed-sequence search as demonstrated in this study based on a validated data set. The availability of open-source search algorithms, such as X!Tandem, makes it feasible to further improve the validation process (manual or automatic) on the basis of "consensus scoring", i.e., the use of multiple (at least two) search algorithms to reduce the number of FPs. complement.

The sample fractionation steps conducted prior to mass detection are critically important for the comprehensive analysis of complex protein mixtures. This paper illustrates the effectiveness of OFFGEL electrophoresis with the Agilent 3100 OFFGEL Fractionator for the fractionation of peptides. An Escherichia coli tryptic digest was separated in 24 fractions, and peptides were identified by reversed-phase liquid chromatography on a microfluidic device with mass spectrometric detection. About 90% of the identified individual peptides were found in only one or two fractions. The distribution of the calculated isoelectric points for the peptides identified in each fraction was especially narrow in the acidic pH range. Standard deviations approached the size of the pH segment covered by the respective fraction. The experimental peptide isoelectric point measured by OFFGEL electrophoresis was used as an additional filter for validation of peptide identifications. Molecular & Cellular Proteomics 5:1968 -1974, 2006.Comprehensive analysis of whole proteomes is an extraordinary challenge because of the complexity and wide range of protein concentrations. The success of proteome analysis projects is highly dependent on the quality of the sample fractionation employed prior to analysis by MS. Reducing sample complexity through efficient fractionation allows more complete in-depth analysis of the sample with MS/MS. For peptide-level analysis, cation-exchange (SCX) 1 and reversedphase chromatography (RP) are typically combined (1). The use of IPG IEF instead of SCX has been described before (2, 3). Compared with cation-exchange chromatography, IPG IEF provides higher resolution separation and experimentally derived pI information, which can be used as a filter criterion for tandem mass spectral data validation (3, 4). However, a major limitation of this method is the tedious post-IEF sample processing that requires cutting the IPG gel strip into sections and then extracting and cleaning up peptides from the gel sections.In the present work, an Escherichia coli total protein digest was fractionated by OFFGEL electrophoresis with the Agilent 3100 OFFGEL Fractionator and by reversed-phase chromatography and then analyzed by mass detection (RP-LC/MS). OFFGEL electrophoresis is a recent advance in separation technology that fractionates proteins or peptides according to their pI (5-8). This technique achieves the same high resolution as IPG gels but recovers the sample in the liquid phase. Therefore, it fits nicely into the LC/MS workflow. After fractionation and acidification, a portion of the sample was injected directly onto a chip-based reversed-phase column without additional sample preparation, eliminating the need for tedious and error-prone peptide isolation from the IPG gel. The distribution of the calculated isoelectric points for the identified peptides in the respective fractions was especially narrow in the acidic pH range, with standard deviations in the range of the expected fraction width. About 90% of the identifie...

A peptide (Manduca sexta ailatostatin) that strongly inhibits juvenile hormone biosynthesis in vitro by the corpora allata from fifth-stadium larvae and adult females has been purified from extracts of heads of pharate adult M. sexta by a nine-step purification procedure. The primary structure of this 15-residue peptide has been determined: pGlu-Val-ArgPhe-Arg-Gln-Cys-Tyr-Phe-Asn-Pro-Ile-Ser-Cys-Phe-OH, where pGlu is pyroglutamate). To our knowledge, this neurohormone has no sequence similarity with any known neuropeptide from other organisms. The synthetic free acid and amide forms showed in vitro activity indistihable from that of native M. sexta aliatostatn. The EDsO of synthetic M. sexta allatostatin on early fifth stadium larval corpora allata in vitro was =2 nM. This inhibition was reversible. In a cross-species study, M. sexta allatostatin also inhibited the corpora allata of adult female Heliothis virescens but had no effect on the activity of corpora allata ofadult females of the beetle Tenebrio molitor, the grasshopper Melanoplus sanguinipes, or the cockroach Periplaneta americana.Juvenile hormone (JH), which is synthesized and released by the retrocerebral corpora allata (CA), plays a vital role in insect development, primarily in the control of metamorphosis, adult sexual maturation, and reproduction (1, 2). Environmental and physiological factors influence neurosecretory centers in the brain that affect the activity of CA through peptidergic materials either transported directly to the CA by axons or released into the blood (3). These neuropeptides may be stimulatory (allatotropins, e.g., refs. 4-8) (27). However, instead of assaying for JH production by chloroform extraction followed by TLC analysis, we used a rapid isooctane partitioning assay for JH (28). In the cross-species studies, we used the same in vitro technique (one or two pair of CA per incubation).Extraction and Preliminary Purification (Steps 14). Thirty thousand trimmed heads of M. sexta (fresh weight, "'.1460 g) were processed in three batches of 10,000. Each batch was defatted by homogenizing in 2 liters of ice-cold acetone and filtered. Residues were extracted with 1.4 liters of 1 M HOAc/20 mM HCO (containing 0.1 mM phenylmethylsulfonyl fluoride and 0.01 mM pepstatin A, prepared immediately before use) and centrifuged at 10,000 x g for 30 min at 40C. The pellet was extracted and centrifuged twice using a total volume of 2.8 liters of the same solution. The combined supernatants were stirred with swollen SP-Sephadex C-25 resin (300 ml) overnight. The resin was allowed to settle for 2 hr, subsequently poured into a Bio-Rad column (50 x 300 mm), and equilibrated with 1 M HOAc. Material was eluted from the column sequentially with 1-liter volumes of 0.05 M NH4OAc (pH 4.0) and 0.05 M, 0.1 M, 0.2 M, 0.4 M, and 0.8 M NH4OAc (pH 7.0). The 0.1 M and 0.2 M fractions, which had M. sexta AS (Mas-AS) activity, were applied directly to 10 g of reversed-phase Vydac C4 packing material (20-30 pAm, contained in a 75-ml polypropylene syr...