Trimethylation Enhancement using Diazomethane (TrEnDi): Rapid On-Column Quaternization of Peptide Amino Groups via Reaction with Diazomethane Significantly Enhances Sensitivity in Mass Spectrometry Analyses via a Fixed, Permanent Positive Charge

2014 ◽  
Vol 86 (7) ◽  
pp. 3291-3299 ◽  
Author(s):  
Karl V. Wasslen ◽  
Le Hoa Tan ◽  
Jeffrey M. Manthorpe ◽  
Jeffrey C. Smith
Keyword(s):  
Mass Spectrometry ◽  
Positive Charge ◽  
Amino Groups

Determination of position of amino groups in aminosugars by mass spectrometry method

1973 ◽  
Vol 22 (12) ◽  
pp. 2713-2716
Author(s):  
A. I. Usov ◽  
R. G. Krylova ◽  
S. N. Ryadovskaya ◽  
V. I. Kadentsev ◽  
O. S. Chizhov
Keyword(s):  
Mass Spectrometry ◽  
Amino Groups ◽  
Spectrometry Method ◽  
Mass Spectrometry Method

scholarly journals The Capsule Sensitizes Streptococcus pneumoniae to α-Defensins Human Neutrophil Proteins 1 to 3

2008 ◽  
Vol 76 (8) ◽  
pp. 3710-3716 ◽  
Author(s):  
Katharina Beiter ◽  
Florian Wartha ◽  
Robert Hurwitz ◽  
Staffan Normark ◽  
Arturo Zychlinsky ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
High Performance Liquid Chromatography ◽  
Streptococcus Pneumoniae ◽  
Human Neutrophil ◽  
High Performance ◽  
Positive Charge ◽  
Early Stage ◽  
Innate Immune ◽  
Intracellular Killing ◽  
Lipoteichoic Acids

ABSTRACT Streptococcus pneumoniae is a major cause of morbidity and mortality worldwide. Its polysaccharide capsule causes resistance to phagocytosis and interferes with the innate immune system's ability to clear infections at an early stage. Nevertheless, we found that encapsulated pneumococci are sensitive to killing by a human neutrophil granule extract. We fractionated the extract by high-performance liquid chromatography and identified α-defensins by mass spectrometry as the proteins responsible for killing pneumococci. Analysis of sensitivity to the commercial α-defensins human neutrophil proteins 1 to 3 (HNP1-3) confirmed these findings. We analyzed the sensitivities of different pneumococcal strains to HNP1-3 and found that encapsulated strains are efficiently killed at physiological concentrations (7.5 μg/ml). Surprisingly, nonencapsulated, nonvirulent pneumococci were significantly less sensitive to α-defensins. The proposed mechanisms of α-defensin resistance in nonencapsulated pneumococci is surface charge modification, e.g., by introduction of positive charge by d-alanylation of surface-exposed lipoteichoic acids. These mechanisms are surmounted by the presence of the capsule, which we hypothesize is masking these charge modifications. Hence, α-defensins in the phagolysosome of neutrophils possibly contribute to intracellular killing after antibody-mediated opsonophagocytosis of encapsulated pneumococci.

scholarly journals A CYTOCHEMICAL STUDY OF CYTOPLASMIC BASIC PROTEINS IN THE ASCIDIAN OOCYTE

1965 ◽  
Vol 25 (2) ◽  
pp. 319-326 ◽  
Author(s):  
Richard Davenport ◽  
Janice C. Davenport
Keyword(s):  
Positive Charge ◽  
Amino Groups ◽  
Acid Dyes ◽  
Fast Green ◽  
Cytochemical Study ◽  
Basic Proteins ◽  
Protein Molecules ◽  
Nucleoprotein Complex ◽  
High Concentrations ◽  
Net Positive Charge

The cytoplasm of young oocytes of the ascidians contains high concentrations of proteins which are stainable with alkaline fast green at pH 8.1 and above. These proteins cannot be stained even with acid dyes at low pH unless RNA is removed. Deamination and formalin blockage of amino groups is incapable of destroying the net positive charge on these protein molecules in the presence of RNA, but these treatments destroy the charge if RNA is removed. It is therefore concluded that basic proteins and RNA exist as a nucleoprotein complex in the ribosomes of these young oocytes. The detectable RNA of the mature oocytes and unfertilized eggs shows no evidence of being associated with basic proteins.

scholarly journals Exploring the Inhibition of CTX-M-9 by β-Lactamase Inhibitors and Carbapenems

2011 ◽  
Vol 55 (7) ◽  
pp. 3465-3475 ◽  
Author(s):  
Christopher R. Bethel ◽  
Magdalena Taracila ◽  
Teresa Shyr ◽  
Jodi M. Thomson ◽  
Anne M. Distler ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Active Site ◽  
Positive Charge ◽  
Evolutionary Strategy ◽  
Ionization Mass ◽  
Ionization Time ◽  
Esi Ms ◽  
Flight Mass Spectrometry ◽  
Before And After ◽  
Enzyme Intermediate

ABSTRACTCurrently, CTX-M β-lactamases are among the most prevalent and most heterogeneous extended-spectrum β-lactamases (ESBLs). In general, CTX-M enzymes are susceptible to inhibition by β-lactamase inhibitors. However, it is unknown if the pathway to inhibition by β-lactamase inhibitors for CTX-M ESBLs is similar to TEM and SHV β-lactamases and why bacteria possessing only CTX-M ESBLs are so susceptible to carbapenems. Here, we have performed a kinetic analysis and timed electrospray ionization mass spectrometry (ESI-MS) studies to reveal the intermediates of inhibition of CTX-M-9, an ESBL representative of this family of enzymes. CTX-M-9 β-lactamase was inactivated by sulbactam, tazobactam, clavulanate, meropenem, doripenem, ertapenem, and a 6-methylidene penem, penem 1.Kivalues ranged from 1.6 ± 0.3 μM (mean ± standard error) for tazobactam to 0.02 ± 0.01 μM for penem 1. Before and after tryptic digestion of the CTX-M-9 β-lactamase apo-enzyme and CTX-M-9 inactivation by inhibitors (meropenem, clavulanate, sulbactam, tazobactam, and penem 1), ESI-MS and matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) identified different adducts attached to the peptide containing the active site Ser70 (+52, 70, 88, and 156 ± 3 atomic mass units). This study shows that a multistep inhibition pathway results from modification or fragmentation with clavulanate, sulbactam, and tazobactam, while a single acyl enzyme intermediate is detected when meropenem and penem 1 inactivate CTX-M-9 β-lactamase. More generally, we propose that Arg276 in CTX-M-9 plays an essential role in the recognition of the C3carboxylate of inhibitors and that the localization of this positive charge to a “region of the active site” rather than a specific residue represents an important evolutionary strategy used by β-lactamases.

scholarly journals Porphyrin–Schiff Base Conjugates Bearing Basic Amino Groups as Antimicrobial Phototherapeutic Agents

Molecules ◽  
2021 ◽  
Vol 26 (19) ◽  
pp. 5877
Author(s):  
María E. Pérez ◽  
Javier E. Durantini ◽  
Eugenia Reynoso ◽  
María G. Alvarez ◽  
María E. Milanesio ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Schiff Base ◽  
Positive Charge ◽  
Aqueous Media ◽  
Fluorescence Emission ◽  
Photodynamic Inactivation ◽  
Type Ii ◽  
Amino Groups ◽  
Absorption Bands ◽  
Photo Oxidation

New porphyrin–Schiff base conjugates bearing one (6) and two (7) basic amino groups were synthesized by condensation between tetrapyrrolic macrocycle-containing amine functions and 4-(3-(N,N-dimethylamino)propoxy)benzaldehyde. This approach allowed us to easily obtain porphyrins substituted by positive charge precursor groups in aqueous media. These compounds showed the typical Soret and four Q absorption bands with red fluorescence emission (ΦF ~ 0.12) in N,N-dimethylformamide. Porphyrins 6 and 7 photosensitized the generation of O2(1Δg) (ΦΔ ~ 0.44) and the photo-oxidation of L-tryptophan. The decomposition of this amino acid was mainly mediated by a type II photoprocess. Moreover, the addition of KI strongly quenched the photodynamic action through a reaction with O2(1Δg) to produce iodine. The photodynamic inactivation capacity induced by porphyrins 6 and 7 was evaluated in Staphylococcus aureus, Escherichia coli, and Candida albicans. Furthermore, the photoinactivation of these microorganisms was improved using potentiation with iodide anions. These porphyrins containing basic aliphatic amino groups can be protonated in biological systems, which provides an amphiphilic character to the tetrapyrrolic macrocycle. This effect allows one to increase the interaction with the cell wall, thus improving photocytotoxic activity against microorganisms.

scholarly journals Derivatization of Aminoglycoside Antibiotics with Tris(2,6-dimethoxyphenyl)carbenium Ion

Acta Naturae ◽  
2016 ◽  
Vol 8 (3) ◽  
pp. 128-135 ◽  
Author(s):  
A. P. Topolyan ◽  
M. A. Belyaeva ◽  
E. E. Bykov ◽  
P. V. Coodan ◽  
E. A. Rogozhin ◽  
...  
Keyword(s):  
Organic Molecules ◽  
Positive Charge ◽  
Aminoglycoside Antibiotics ◽  
Maldi Ms ◽  
Amino Groups ◽  
Uv Detectors ◽  
Aromatic Residue ◽  
Amide Bonds ◽  
Carbenium Ion

Detection of aminoglycoside antibiotics by MS or HPLC is complicated, because a) carbohydrate molecules have low ionization ability in comparison with other organic molecules (particularly in MALDI-MS), and b) the lack of aromatics and/or amide bonds in the molecules makes common HPLC UV-detectors useless. Here, we report on the application of a previously developed method for amine derivatization with tris(2,6-dimethoxyphenyl)carbenium ion to selective modification of aminoglycoside antibiotics. Only amino groups bound to primary carbons get modified. The attached aromatic residue carries a permanent positive charge. This makes it easy to detect aminoglycoside antibiotics by MS-methods and HPLC, both as individual compounds and in mixtures.

A Diagonal Paper Electrophoretic Method for the Selective Isolation of Histidyl Peptides

1971 ◽  
Vol 49 (11) ◽  
pp. 1225-1232 ◽  
Author(s):  
W. H. Cruickshank ◽  
T. M. Radhakrishnan ◽  
H. Kaplan
Keyword(s):  
High Voltage ◽  
Positive Charge ◽  
Proteolytic Enzymes ◽  
Paper Electrophoresis ◽  
Selective Isolation ◽  
Amino Groups ◽  
Electrophoretic Method ◽  
Original Direction ◽  
Citraconic Anhydride ◽  
Net Positive Charge

Thiolysis of an imidazolyl-dinitrophenyl-histidyl peptide at either pH 3.5 or 6.5 results in an increase in the net positive charge on the peptide. It is shown that this property can be used to form the basis of a diagonal paper electrophoretic purification of histidyl peptides from proteins. The amino groups of the protein are first reacted with citraconic anhydride and then the citraconyl protein is reacted with 1-fluoro-2,4-dinitrobenzene. The dinitrophenyl-citraconyl protein is digested with pepsin in 10% formic acid and, if necessary, with other proteolytic enzymes. The enzymatic digest is subjected to high-voltage paper electrophoresis at either pH 3.5 or 6.5. A guide strip is removed, thiolyzed with 2-mercaptoethanol, and subjected to electrophoresis at the same pH at right angles to the original direction of electrophoresis. The histidyl peptides are displaced off the diagonal toward the cathode. The off-diagonal peptides are isolated from the original electrophoretogram by thiolysis and electrophoresis using the diagonal electrophoretogram to locate the positions of the dinitrophenyl-histidyl peptides.

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