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1.
J Am Soc Mass Spectrom ; 31(9): 1896-1902, 2020 Sep 02.
Article in English | MEDLINE | ID: mdl-32799534

ABSTRACT

Top-down proteomics by mass spectrometry (MS) involves the mass measurement of an intact protein followed by subsequent activation of the protein to generate product ions. Electron-based fragmentation methods like electron capture dissociation and electron transfer dissociation are widely used for these types of analyses. Recently, electron ionization dissociation (EID), which utilizes higher energy electrons (>20 eV) has been suggested to be more efficient for top-down protein fragmentation compared to other electron-based dissociation methods. Here, we demonstrate that the use of EID enhances protein fragmentation and subsequent detection of protein fragments. Protein product ions can form by either single cleavage events, resulting in terminal fragments containing the C-terminus or N-terminus of the protein, or by multiple cleavage events to give rise to internal fragments that include neither the C-terminus nor the N-terminus of the protein. Conventionally, internal fragments have been disregarded, as reliable assignments of these fragments were limited. Here, we demonstrate that internal fragments generated by EID can account for ∼20-40% of the mass spectral signals detected by top-down EID-MS experiments. By including internal fragments, the extent of the protein sequence that can be explained from a single tandem mass spectrum increases from ∼50 to ∼99% for 29 kDa carbonic anhydrase II and 8.6 kDa ubiquitin. When searching for internal fragments during data analysis, previously unassigned peaks can be readily and accurately assigned to confirm a given protein sequence and to enhance the utility of top-down protein sequencing experiments.


Subject(s)
Mass Spectrometry/methods , Peptide Fragments/chemistry , Proteins/chemistry , Proteomics/methods , Animals , Ions/analysis , Ions/chemistry , Peptide Fragments/analysis , Proteins/analysis , Sequence Analysis, Protein
2.
PLoS One ; 11(6): e0157126, 2016.
Article in English | MEDLINE | ID: mdl-27280889

ABSTRACT

Several lymphatic reporter mouse lines have recently been developed to significantly improve imaging of lymphatic vessels. Nonetheless, the usage of direct visualization of lymphatic vessels has not been fully explored and documented. Here, we characterized a new Prox1-tdTomato transgenic lymphatic reporter mouse line, and demonstrated how this animal tool enables the researchers to efficiently assess developmental, surgical and pathological lymphangiogenesis by direct visualization of lymphatic vessels. Moreover, we have derived embryonic stem cells from this reporter line, and successfully differentiated them into lymphatic vessels in vivo. In conclusion, these experimental tools and techniques will help advance lymphatic research.


Subject(s)
Embryonic Stem Cells/cytology , Lymphangiogenesis/physiology , Lymphatic Vessels/pathology , Animals , Genes, Reporter , Lymphatic Vessels/surgery , Mice , Mice, Inbred NOD , Mice, Nude , Mice, SCID , Mice, Transgenic , Models, Animal
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