Molecular complexity of the major urinary protein system of the Norway rat, Rattus norvegicus

ABSTRACTMajor urinary proteins (MUP) are the major component of the urinary protein fraction in house mice (Mus spp.) and rats (Rattus spp.). The structure, polymorphism and functions of these lipocalins have been well described in the western European house mouse (Mus musculus domesticus), clarifying their role in semiochemical communication. The complexity of these roles in the mouse raises the question of similar functions in other rodents, including the Norway rat, Rattus norvegicus. Norway rats express MUPs in urine but information about specific MUP isoform sequences and functions is limited. In this study, we present a detailed molecular characterization of the MUP proteoforms expressed in the urine of two laboratory strains, Wistar Han and Brown Norway, and wild caught animals, using a combination of manual gene annotation, intact protein mass spectrometry and bottom-up mass spectrometry-based proteomic approaches. Detailed sequencing of the proteins reveals a less complex pattern of primary sequence polymorphism than the mouse. However, unlike the mouse, rat MUPs exhibit added complexity in the form of post-translational modifications including phosphorylation and exoproteolytic trimming of specific isoforms. The possibility that urinary MUPs may have different roles in rat chemical communication than those they play in the house mouse is also discussed.

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Intact Protein Mass Spectrometry for Therapeutic Protein Quantitation, Pharmacokinetics, and Biotransformation in Preclinical and Clinical Studies: An Industry Perspective

Journal of the American Society for Mass Spectrometry ◽

10.1021/jasms.0c00270 ◽

2020 ◽

Author(s):

John F. Kellie ◽

John C. Tran ◽

Wenying Jian ◽

Barry Jones ◽

John T. Mehl ◽

...

Keyword(s):

Mass Spectrometry ◽

Clinical Studies ◽

Therapeutic Protein ◽

Intact Protein ◽

Protein Mass ◽

Protein Mass Spectrometry ◽

Protein Quantitation ◽

Preclinical And Clinical Studies

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Protein mass measurement combined with mass spectrometric sequencing of protein digests for detection and characterization of protein modifications1

Canadian Journal of Chemistry ◽

10.1139/v06-114 ◽

2006 ◽

Vol 84 (7) ◽

pp. 986-997 ◽

Cited By ~ 2

Author(s):

Chengjie Ji ◽

Zhengping Wang ◽

Liang Li

Keyword(s):

Mass Spectrometry ◽

Gel Electrophoresis ◽

Protein Modification ◽

Mass Spectrometric ◽

Maldi Ms ◽

Intact Protein ◽

Protein Mass ◽

Cell Extracts ◽

Peptide Mass

A method for the characterization of modifications of low molecular weight proteins (<20 kDa) extracted from a microorganism based on the use of multiple separation tools and mass spectrometric techniques is described. In this method, intact proteins from cell extracts are first separated and fractionated by liquid chromatography (LC). Individual fractions are then analyzed by matrix-assisted laser desorption ionization (MALDI) mass spectrometry (MS) to provide intact protein mass information. The fractions are further characterized by using trypsin digestion and LC electrospray ionization (ESI) MS/MS analysis of the resultant peptides to identify the proteins. Gel electrophoresis of a fraction is also carried out to estimate the molecular masses of the proteins. The gel bands are identified by in-gel digestion and peptide mass mapping and sequencing using MALDI-MS and MALDI-MS/MS. The combined information generated from these experiments is interpreted for detecting and characterizing modified proteins. This method has been developed and applied to the analysis of posttranslational modifications (PTMs) of low-mass proteins (5–20 kDa) extracted from a relatively well-characterized microorganism, Escherichia coli. Using this method, not only previously reported PTMs involving acetylation, methylation, oxidation, and the removal of signal peptides, but also two novel PTMs, namely loss of N-terminal Met-Thr-Met (MTM) and hydroxylation of arginine, were identified. It is envisaged that this method should be applicable to other relatively simple microorganisms for the discovery of new PTMs.Key words: top-down proteomics, protein modification, HPLC, gel electrophoresis, tandem mass spectrometry.

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Rapid Fingerprinting of Milk Thermal Processing History by Intact Protein Mass Spectrometry with Nondenaturing Chromatography

Journal of Agricultural and Food Chemistry ◽

10.1021/jf203151e ◽

2011 ◽

Vol 59 (23) ◽

pp. 12420-12427 ◽

Cited By ~ 22

Author(s):

Phil Johnson ◽

Mark Philo ◽

Andrew Watson ◽

E. N. Clare Mills

Keyword(s):

Mass Spectrometry ◽

Thermal Processing ◽

Intact Protein ◽

Protein Mass ◽

Protein Mass Spectrometry ◽

Processing History

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Mass spectrometry for structural analysis and quantification of the Major Urinary Proteins of the house mouse

International Journal of Mass Spectrometry ◽

10.1016/j.ijms.2015.07.026 ◽

2015 ◽

Vol 391 ◽

pp. 146-156 ◽

Cited By ~ 9

Author(s):

Robert J. Beynon ◽

Stuart D. Armstrong ◽

Amy J. Claydon ◽

Amanda J. Davidson ◽

Claire E. Eyers ◽

...

Keyword(s):

Mass Spectrometry ◽

Structural Analysis ◽

House Mouse ◽

Urinary Proteins ◽

Major Urinary Proteins

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Top-Down and Intact Protein Mass Spectrometry Data Visualization for Proteoform Analysis Using VisioProt-MS

Bioinformatics and Biology Insights ◽

10.1177/1177932219868223 ◽

2019 ◽

Vol 13 ◽

pp. 117793221986822 ◽

Cited By ~ 3

Author(s):

Jean Lesne ◽

Marie-Pierre Bousquet ◽

Julien Marcoux ◽

Marie Locard-Paulet

Keyword(s):

Mass Spectrometry ◽

Liquid Chromatography ◽

Data Visualization ◽

Web Application ◽

Three Dimensional ◽

Mass Spectrometry Data ◽

Intact Protein ◽

Top Down ◽

Molecular Weights ◽

Protein Mass

The rise of intact protein analysis by mass spectrometry (MS) was accompanied by an increasing need for flexible tools allowing data visualization and analysis. These include inspection of the deconvoluted molecular weights of the proteoforms eluted alongside liquid chromatography (LC) through their representation in three-dimensional (3D) liquid chromatography coupled to mass spectrometry (LC-MS) maps (plots of deconvoluted molecular weights, retention times, and intensity of the MS signal). With this aim, we developed a free and open-source web application named VisioProt-MS ( https://masstools.ipbs.fr/mstools/visioprot-ms/ ). VisioProt-MS is highly compatible with many algorithms and software developed by the community to integrate and deconvolute top-down and intact protein MS data. Its dynamic and user-friendly features greatly facilitate analysis through several graphical representations dedicated to MS and tandem mass spectrometry (MS/MS) analysis of proteoforms in complex samples. Here, we will illustrate the importance of LC-MS map visualization to optimize top-down acquisition/search parameters and analyze intact protein MS data. We will go through the main features of VisioProt-MS using the human proteasomal 20S core particle as a user-case.

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