Lázaro Betancourt

Activation of Rho through a cross-link with polyamines catalyzed by Bordetella dermonecrotizing toxin

The small GTPase Rho, which regulates a variety of cell functions, also serves as a specific substrate for bacterial toxins. Here we demonstrate that Bordetella dermonecrotizing toxin (DNT) catalyzes cross‐linking of Rho with ubiquitous polyamines such as putrescine, spermidine and spermine. Mass spectrometric analyses revealed that the cross‐link occurred at Gln63, which had been reported to be deamidated by DNT in the absence of polyamines. Rac1 and Cdc42, other members of the Rho family GTPases, were also polyaminated by DNT. The polyamination, like the deamidation, markedly reduced the GTPase activity of Rho without affecting its GTP‐binding activity, indicating that polyaminated Rho behaves as a constitutively active analog. Moreover, polyamine‐linked Rho, even in the GDP‐bound form, associated more effectively with its effector ROCK than deamidated Rho in the GTP‐bound form and, when microinjected into cells, induced the anomalous formation of stress fibers indistinguishable from those seen in DNT‐treated cells. The results imply that the polyamine‐linked Rho, transducing signals to downstream ROCK in a novel GTP‐independent manner, plays an important role in DNT cell toxicity.

Automated interpretation of low-energy collision-induced dissociation spectra by SeqMS, a software aid for de novo sequencing by tandem mass spectrometry

SeqMS, a software aid for de novo sequencing by tandem mass spectrometry (MS/MS), which was initially developed for the automated interpretation of high‐energy collision‐induced dissociation (CID) MS/MS spectra of peptides, has been applied to the interpretation of low‐energy CID and post‐source decay (PSD) spectra of peptides. Based on peptide backbone fragmented ions and their related ions, which are the dominant ions observed in the latter two techniques, the types of ions and their propensities to be observed have been optimized for efficient interpretation of the spectra. In a typical example, the modified SeqMS allowed the complete sequencing of a 31‐amino acid synthetic peptide, except for the isobaric amino acids (Leu or Ile, and Lys or Gln), based on only the low‐energy CID‐MS/MS spectrum.

Automated interpretation of high-energy collision-induced dissociation spectra of singly protonated peptides by ‘seqms', a software aid for de novo sequencing by tandem mass spectrometry

SeqMS, a software program designed for the automated interpretation of high-energy collision-induced dissociation (CID) mass spectra of singly protonated peptides ionized by fast atom bombardment, has been developed. The software is capable of probing the sequence of an unknown peptide, and even of certain modified peptides. The program, compiled for WINDOWS95 or NT, also permits the retrieval of raw data and the reconstruction of the spectra on a user-friendly graphical interface with the aid of several tools for processing the spectra, which include setting multiple threshold levels and automatic peak detection. SeqMS is capable of generating candidate sequences, based on the detected peaks, and of displaying the resulting assignments for each candidate in a spectrum or in tabular form. The software has the following capabilities: 1) the ions derived from backbone and side-chain fragmentations, internal and immonium ions, and side-chain loss ions can be used for calculation; 2) 18O-labeling of a peptide at the C terminus, a methodology which was developed to differentiate N-terminal from C-terminal ions, is applicable as an optional setting; 3) modified amino acids and N- or C-terminal blocking groups are taken into account for calculation according to the users setting in a library; 4) amino acid composition and partial or complete amino acid sequence of a peptide can be used as input for calculation; 5) the assignments of signal output in a spectrum can be graphically edited, and then re-calculated based on the edited peaks. The efficacy of the program is demonstrated by testing 74 high-energy CID spectra, obtained using a four-sector instrument, of synthetic, proteolytic, and biologically active peptides, some of which contain modified groups.

CIGB-300, a synthetic peptide-based drug that targets the CK2 phosphoaceptor domain. Translational and clinical research

CK2 represents an oncology target scientifically validated. However, clinical research with inhibitors of the CK2-mediated phosphorylation event is still insufficient to recognize it as a clinically validated target. CIGB-300, an investigational peptide-based drug that targets the phosphoaceptor site, binds to a CK2 substrate array in vitro but mainly to B23/nucleophosmin in vivo. The CIGB-300 proapoptotic effect is preceded by its nucleolar localization, inhibition of the CK2-mediated phosphorylation on B23/nucleophosmin and nucleolar disassembly. Importantly, CIGB-300 shifted a protein array linked to apoptosis, ribosome biogenesis, cell proliferation, glycolisis, and cell motility in proteomic studies which helped to understand its mechanism of action. In the clinical ground, CIGB-300 has proved to be safe and well tolerated in a First-in-Human trial in women with cervical malignancies who also experienced signs of clinical benefit. In a second Phase 1 clinical trial in women with cervical cancer stage IB2/II, the MTD and DLT have been also identified in the clinical setting. Interestingly, in cervical tumors the B23/nucleophosmin protein levels were significantly reduced after CIGB-300 treatment at the nucleus compartment. In addition, expanded use of CIGB-300 in case studies has evidenced antitumor activity when administered as compassional option. Collectively, our data outline important clues on translational and clinical research from this novel peptide-based drug reinforcing its perspectives to treat cancer and paving the way to validate CK2 as a promising target in oncology.

In silico analysis of accurate proteomics, complemented by selective isolation of peptides

Protein identification by mass spectrometry is mainly based on MS/MS spectra and the accuracy of molecular mass determination. However, the high complexity and dynamic ranges for any species of proteomic samples, surpass the separation capacity and detection power of the most advanced multidimensional liquid chromatographs and mass spectrometers. Only a tiny portion of signals is selected for MS/MS experiments and a still considerable number of them do not provide reliable peptide identification. In this article, an in silico analysis for a novel methodology of peptides and proteins identification is described. The approach is based on mass accuracy, isoelectric point (pI), retention time (t(R)) and N-terminal amino acid determination as protein identification criteria regardless of high quality MS/MS spectra. When the methodology was combined with the selective isolation methods, the number of unique peptides and identified proteins increases. Finally, to demonstrate the feasibility of the methodology, an OFFGEL-LC-MS/MS experiment was also implemented. We compared the more reliable peptide identified with MS/MS information, and peptide identified with three experimental features (pI, t(R), molecular mass). Also, two theoretical assumptions from MS/MS identification (selective isolation of peptides and N-terminal amino acid) were analyzed. Our results show that using the information provided by these features and selective isolation methods we could found the 93% of the high confidence protein identified by MS/MS with false-positive rate lower than 5%.

Development and validation of a bioanalytical LC-MS method for the quantification of GHRP-6 in human plasma.

Growth hormone-releasing peptide 6 (GHRP-6, His-(DTrp)-Ala-Trp-(DPhe)-Lys-NH₂, MW=872.44 Da) is a potent growth hormone secretagogue that exhibits a cytoprotective effect, maintaining tissue viability during acute ischemia/reperfusion episodes in different organs like small bowel, liver and kidneys. In the present work a quantitative method to analyze GHRP-6 in human plasma was developed and fully validated following FDA guidelines. The method uses an internal standard (IS) of GHRP-6 with ¹³C-labeled Alanine for quantification. Sample processing includes a precipitation step with cold acetone to remove the most abundant plasma proteins, recovering the GHRP-6 peptide with a high yield. Quantification was achieved by LC-MS in positive full scan mode in a Q-Tof mass spectrometer. The sensitivity of the method was evaluated, establishing the lower limit of quantification at 5 ng/mL and a range for the calibration curve from 5 ng/mL to 50 ng/mL. A dilution integrity test was performed to analyze samples at higher concentration of GHRP-6. The validation process involved five calibration curves and the analysis of quality control samples to determine accuracy and precision. The calibration curves showed R² higher than 0.988. The stability of the analyte and its internal standard (IS) was demonstrated in all conditions the samples would experience in a real time analyses. This method was applied to the quantification of GHRP-6 in plasma from nine healthy volunteers participating in a phase I clinical trial.

Levansucrase from Acetobacter diazotrophicus SRT4 Is Secreted via Periplasm by a Signal-Peptide-Dependent Pathway

Abstract.Acetobacter diazotrophicus SRT4 secretes a constitutive levansucrase (LsdA) (EC 2.4.1.10) that is responsible for sucrose utilization. Immunogold electron microscopical studies revealed that LsdA accumulates in the periplasm before secretion. The periplasmic and extracellular forms of the enzyme were purified to homogeneity. Both proteins exhibited similar physical and biochemical characteristics indicating that LsdA adopts its final conformation in the periplasm. The N-terminal sequence of mature LsdA was pGlu-Gly-Asn-Phe-Ser-Arg as determined by PSD-MALDI-TOFMS (post-source decay—matrix-assisted laser desorption/ionization—time-of-flight mass spectrometry). Comparison of this sequence with the predicted precursor protein revealed the cleavage of a 30-residue typical signal peptide followed by the formation of the pyroglutamic acid (pGlu) residue. Thus, in contrast with other Gram-negative bacteria, A. diazotrophicus secretes levansucrase by a signal-peptide-dependent mechanism.

Isoelectric point optimization using peptide descriptors and support vector machines.

IPG (Immobilized pH Gradient) based separations are frequently used as the first step in shotgun proteomics methods; it yields an increase in both the dynamic range and resolution of peptide separation prior to the LC-MS analysis. Experimental isoelectric point (pI) values can improve peptide identifications in conjunction with MS/MS information. Thus, accurate estimation of the pI value based on the amino acid sequence becomes critical to perform these kinds of experiments. Nowadays, pI is commonly predicted using the charge-state model [1], and/or the cofactor algorithm [2]. However, none of these methods is capable of calculating the pI value for basic peptides accurately. In this manuscript, we present an new approach that can significant improve the pI estimation, by using Support Vector Machines (SVM) [3], an experimental amino acid descriptor taken from the AAIndex database [4] and the isoelectric point predicted by the charge-state model. Our results have shown a strong correlation (R(2)=0.98) between the predicted and observed values, with a standard deviation of 0.32 pH units across the complete pH range.

Proteomic Profile Regulated by the Anticancer Peptide CIGB-300 in Non-Small Cell Lung Cancer (NSCLC) Cells

CIGB-300 is a proapoptotic peptide-based drug that abrogates the CK2-mediated phosphorylation. This peptide has antineoplastic effect on lung cancer cells in vitro and in vivo. To understand the mechanisms involved on such anticancer activity, the NCI-H125 cell line proteomic profile after short-term incubation (45 min) with CIGB-300 was investigated. As determined by 2-DE or 2D-LC-MS/MS, 137 proteins changed their abundances more than 2-fold in response to the CIGB-300 treatment. The expression levels of proteins related to ribosome biogenesis, metastasis, cell survival and proliferation, apoptosis, and drug resistance were significantly modulated by the presence of CIGB-300. The protein translation process was the most affected (23% of the identified proteins). From the proteome analysis of the NCI-H125 cell line, novel potentialities for CIGB-300 as anticancer agent were evidenced.

Proteomic study via a non-gel based approach of meningococcal outer membrane vesicle vaccine obtained from strain CU385: A road map for discovering new antigens

This work presents the results from a study of the protein composition of outer membrane vesicles from VA-MENGOC-BC® (Finlay Institute, Cuba), an available vaccine against serogroup B Neisseria meningitidis. Proteins were identified by means of SCAPE, a 2DE-free method for proteome studies. More than one hundred proteins were detected by tandem liquid chromatography-mass spectrometry analysis of fractions enriched in peptides devoid of histidine or arginine residues, providing a detailed description of the vaccine. A bioinformatic analysis of the identified components resulted in the identification of 31 outer membrane proteins and three conserved hypothetical proteins, allowing the cloning, expression, purification and immunological study of two of them (NMB0088 and NMB1796) as new antigens.