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1.
Chembiochem ; 18(12): 1117-1122, 2017 06 19.
Article in English | MEDLINE | ID: mdl-28425166

ABSTRACT

The uranyl ion (UO22+ ) binds phosphopeptides with high affinity, and when irradiated with UV-light, it can cleave the peptide backbone. In this study, high-accuracy tandem mass spectrometry and enzymatic assays were used to characterise the photocleavage products resulting from the uranyl photocleavage reaction of a tetraphosphorylated ß-casein model peptide. We show that the primary photocleavage products of the uranyl-catalysed reaction are C-terminally amidated. This could be of great interest to the pharmaceutical industry, as efficient peptide amidation reactions are one of the top challenges in green pharmaceutical chemistry.


Subject(s)
Amides/chemistry , Caseins/chemistry , Phosphopeptides/chemistry , Uranium Compounds/chemistry , Amino Acid Sequence , Carboxypeptidases/chemistry , Caseins/radiation effects , Cations, Divalent , Enzyme Assays , Green Chemistry Technology , Phosphopeptides/radiation effects , Photolysis , Protein Binding , Tandem Mass Spectrometry , Ultraviolet Rays
2.
J Am Soc Mass Spectrom ; 24(11): 1623-33, 2013 Nov.
Article in English | MEDLINE | ID: mdl-23677544

ABSTRACT

We describe the implementation and characterization of activated ion electron transfer dissociation (AI-ETD) on a hybrid QLT-Orbitrap mass spectrometer. AI-ETD was performed using a collision cell that was modified to enable ETD reactions, in addition to normal collisional activation. The instrument manifold was modified to enable irradiation of ions along the axis of this modified cell with IR photons from a CO2 laser. Laser power settings were optimized for both charge (z) and mass to charge (m/z) and the instrument control firmware was updated to allow for automated adjustments to the level of irradiation. This implementation of AI-ETD yielded 1.6-fold more unique identifications than ETD in an nLC-MS/MS analysis of tryptic yeast peptides. Furthermore, we investigated the application of AI-ETD on large scale analysis of phosphopeptides, where laser power aids ETD, but can produce b- and y-type ions because of the phosphoryl moiety's high IR adsorption. nLC-MS/MS analysis of phosphopeptides derived from human embryonic stem cells using AI-ETD yielded 2.4-fold more unique identifications than ETD alone, demonstrating a promising advance in ETD sequencing of PTM containing peptides.


Subject(s)
Electrons , Ions/chemistry , Mass Spectrometry/instrumentation , Phosphopeptides/analysis , Embryonic Stem Cells/chemistry , Humans , Infrared Rays , Lasers , Peptides/radiation effects , Phosphopeptides/radiation effects , Tandem Mass Spectrometry
3.
J Am Soc Mass Spectrom ; 20(12): 2334-41, 2009 Dec.
Article in English | MEDLINE | ID: mdl-19819166

ABSTRACT

Vacuum UV photodissociation tandem mass spectra of singly charged arginine-terminated phosphopeptides were recorded at times ranging from 300 ns to ms after photoexcitation, to investigate when the phosphate group falls off from the precursor and product ions and whether loss of phosphate can be eliminated in tandem mass spectra. For peptide ions containing phosphoserine and phosphothreonine, little loss of 98 Da from the product ions was observed up to 1 micros after photoexcitation. However, neutral losses from the precursor ions were considerable just 300 ns after photoactivation. Loss of 98 Da from product ions first appears about 1 micros after laser irradiation and becomes more common 13 micros after photoexcitation. Consistent with previous reports, phosphotyrosine was more stable than either phosphoserine or phosphothreonine.


Subject(s)
Models, Chemical , Phosphopeptides/chemistry , Phosphopeptides/radiation effects , Spectrometry, Mass, Electrospray Ionization/methods , Ions/radiation effects , Phosphopeptides/analysis , Protons , Radiation Dosage , Ultraviolet Rays
4.
Rapid Commun Mass Spectrom ; 20(3): 507-11, 2006.
Article in English | MEDLINE | ID: mdl-16402344

ABSTRACT

Protonated precursor ions of phosphorylated peptides containing a tyrosyl residue have been subjected to UV laser-induced dissociation (LID) at a wavelength of 220 nm and to collision-induced dissociation (CID) in an ion trap. As expected, neutral loss of the phosphate group is one of the predominant fragmentation channels during CID together with H2O elimination. In contrast, LID leads mainly to the homolytic cleavage of the tyrosyl side chain and a restrained loss of the phosphate group. Interestingly, the intensity of the dephosphorylated fragment ion is greatly minimized when CID is carried out next on the radical precursor ion of the singly and doubly charged species.


Subject(s)
Phosphopeptides/chemistry , Phosphopeptides/radiation effects , Phosphoserine/chemistry , Ultraviolet Rays , Amino Acid Sequence , Lasers , Mass Spectrometry , Molecular Sequence Data , Phosphorylation/radiation effects , Photochemistry
5.
Nat Biotechnol ; 22(8): 993-1000, 2004 Aug.
Article in English | MEDLINE | ID: mdl-15273693

ABSTRACT

Using classical genetics to study modular phosphopeptide-binding domains within a family of proteins that are functionally redundant is difficult when other members of the domain family compensate for the product of the knocked-out gene. Here we describe a chemical genetics approach that overcomes this limitation by using UV-activatable caged phosphopeptides. By incorporating a caged phosphoserine residue within a consensus motif, these reagents simultaneously and synchronously inactivate all phosphoserine/phosphothreonine-binding domain family members in a rapid and temporally regulated manner. We applied this approach to study the global function of 14-3-3 proteins in cell cycle control. Activation of the caged phosphopeptides by UV irradiation displaced endogenous proteins from 14-3-3-binding, causing premature cell cycle entry, release of G1 cells from interphase arrest and loss of the S-phase checkpoint after DNA damage, accompanied by high levels of cell death. This class of reagents will greatly facilitate molecular dissection of kinase-dependent signaling pathways when applied to other phosphopeptide-binding domains including SH2, Polo-box and tandem BRCT domains.


Subject(s)
14-3-3 Proteins/metabolism , G1 Phase , Osteosarcoma/metabolism , Osteosarcoma/pathology , Phosphopeptides/metabolism , Photochemistry/methods , S Phase , Apoptosis , Cell Line, Tumor , Cell Survival , Humans , Phosphopeptides/radiation effects , Recombinant Proteins/metabolism , Recombinant Proteins/radiation effects , Signal Transduction , Structure-Activity Relationship , Time Factors , Ultraviolet Rays
6.
Z Ernahrungswiss ; 30(3): 227-32, 1991 Sep.
Article in English | MEDLINE | ID: mdl-1763559

ABSTRACT

Phosphopeptides derived from casein may function as carriers for calcium and trace elements. In regard to such specific nutritive effects, the heat-induced changes in tryptic phosphopeptides liberated from bovine sodium caseinate as a model system were investigated. Both microwave and oven heating resulted in a marked loss of peptide-bound phosphorous (dephosphorylation) and a decrease of casein-phosphopeptides in the soluble part of the tryptic hydrolysate. It is concluded that hydrolysis of phosphoseryl to seryl residues was the prevailing degradation step to soluble proteolytic products, whereas lysinoalanyl-casein is claimed to be present almost exclusively in the pH 4.6-insoluble part of the tryptic digest.


Subject(s)
Caseins/chemistry , Hot Temperature , Microwaves , Phosphopeptides/metabolism , Animals , Caseins/radiation effects , Cattle , Chromatography, High Pressure Liquid , Hydrogen-Ion Concentration , Phosphopeptides/radiation effects
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