2D SDS PAGE in Combination with Western Blotting and Mass Spectrometry Is a Robust Method for Protein Analysis with Many Applications

Purpose. To investigate the effect of ocular hypertension-induced isomerization of aspartic acid in retinal proteins. Methods. Adult Wistar rats with ocular hypertension were used as an experimental model. D-β-aspartic acid-containing proteins were isolated by SDS-PAGE and western blot with an anti-D-β-aspartic acid antibody and identified by liquid chromatography-mass spectrometry analysis. The concentration of ATP was measured by ELISA. Results. D-β-aspartic acid was expressed in a protein band at around 44.5 kDa at much higher quantities in the retinas of rats with ocular hypertension than in those of normotensive rats. The 44.5 kDa protein band was mainly composed of α-enolase, S-arrestin, and ATP synthase subunits α and β, in both the ocular hypertensive and normotensive retinas. Moreover, increasing intraocular pressure was correlated with increasing ATP concentrations in the retinas of rats. Conclusion. Ocular hypertension affected the expression of proteins containing D-β-aspartic acid, including ATP synthase subunits, and up-regulation of ATP in the retinas of rats.

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Development of a New Highly Selective Monoclonal Antibody against Preferentially Expressed Antigen in Melanoma (PRAME) and Identification of the Target Epitope by Bio-Layer Interferometry

International Journal of Molecular Sciences ◽

10.3390/ijms22063166 ◽

2021 ◽

Vol 22 (6) ◽

pp. 3166

Author(s):

Jwala Priyadarsini Sivaccumar ◽

Antonio Leonardi ◽

Emanuela Iaccarino ◽

Giusy Corvino ◽

Luca Sanguigno ◽

...

Keyword(s):

Mass Spectrometry ◽

Monoclonal Antibody ◽

Monoclonal Antibodies ◽

Western Blotting ◽

Cancer Biomarkers ◽

Trypsin Digestion ◽

Protein Fragments ◽

Target Epitope ◽

Potential Activity ◽

Antibody Epitopes

Background: Monoclonal antibodies (mAbs) against cancer biomarkers are key reagents in diagnosis and therapy. One such relevant biomarker is a preferentially expressed antigen in melanoma (PRAME) that is selectively expressed in many tumors. Knowing mAb’s epitope is of utmost importance for understanding the potential activity and therapeutic prospective of the reagents. Methods: We generated a mAb against PRAME immunizing mice with PRAME fragment 161–415; the affinity of the antibody for the protein was evaluated by ELISA and SPR, and its ability to detect the protein in cells was probed by cytofluorimetry and Western blotting experiments. The antibody epitope was identified immobilizing the mAb on bio-layer interferometry (BLI) sensor chip, capturing protein fragments obtained following trypsin digestion and performing mass spectrometry analyses. Results: A mAb against PRAME with an affinity of 35 pM was obtained and characterized. Its epitope on PRAME was localized on residues 202–212, taking advantage of the low volumes and lack of fluidics underlying the BLI settings. Conclusions: The new anti-PRAME mAb recognizes the folded protein on the surface of cell membranes suggesting that the antibody’s epitope is well exposed. BLI sensor chips can be used to identify antibody epitopes.

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Synthesis and Matrix Properties of α-Cyano-5-phenyl-2,4-pentadienic Acid (CPPA) for Intact Proteins Analysis by Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry

Molecules ◽

10.3390/molecules25246054 ◽

2020 ◽

Vol 25 (24) ◽

pp. 6054

Author(s):

Antonio Monopoli ◽

Angelo Nacci ◽

Tommaso R. I. Cataldi ◽

Cosima D. Calvano

Keyword(s):

Mass Spectrometry ◽

Laser Desorption ◽

Protein Analysis ◽

Ionization Mass Spectrometry ◽

Intact Protein ◽

Ionization Mass ◽

Laser Desorption Ionization ◽

Desorption Ionization ◽

Matrix Assisted Laser ◽

Matrix Assisted Laser Desorption

The effectiveness of a synthesized matrix, α-cyano-5-phenyl-2,4-pentadienic acid (CPPA), for protein analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) in complex samples such as foodstuff and bacterial extracts, is demonstrated. Ultraviolet (UV) absorption along with laser desorption/ionization mass spectrometry (LDI-MS) experiments were systematically conducted in positive ion mode under standard Nd:YLF laser excitation with the aim of characterizing the matrix in terms of wavelength absorption and proton affinity. Besides, the results for standard proteins revealed that CPPA significantly enhanced the protein signals, reduced the spot-to-spot variability and increased the spot homogeneity. The CPPA matrix was successful employed to investigate intact microorganisms, milk and seed extracts for protein profiling. Compared to conventional matrices such as sinapinic acid (SA), α-cyano-4-hydroxycinnamic acid (CHCA) and 4-chloro-α-cyanocinnamic acid (CClCA), CPPA exhibited better signal-to-noise (S/N) ratios and a uniform response for most examined proteins occurring in milk, hazelnut and in intact bacterial cells of E. coli. These findings not only provide a reactive proton transfer MALDI matrix with excellent reproducibility and sensitivity, but also contribute to extending the battery of useful matrices for intact protein analysis.

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Protein Analysis by Electrospray-Orbital Frequency Analyzer with Charge Detection Mass Spectrometry Algorithm

Journal of the American Society for Mass Spectrometry ◽

10.1021/jasms.0c00445 ◽

2021 ◽

Author(s):

Aleksandr Rusinov ◽

Li Ding ◽

Sergey Smirnov ◽

Patrick Knight ◽

Roch Andrzejewski ◽

...

Keyword(s):

Mass Spectrometry ◽

Protein Analysis ◽

Charge Detection Mass Spectrometry ◽

Charge Detection ◽

Orbital Frequency

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Application of 2D BN/SDS-PAGE coupled with mass spectrometry for identification of VDAC-associated protein complexes related to mitochondrial binding sites for type I brain hexokinase

Mitochondrion ◽

10.1016/j.mito.2013.05.009 ◽

2013 ◽

Vol 13 (6) ◽

pp. 823-830 ◽

Cited By ~ 1

Author(s):

Carla Rossini Crepaldi ◽

Phelipe Augusto Mariano Vitale ◽

Andrea Cristina Tesch ◽

Hélen Julie Laure ◽

José César Rosa ◽

...

Keyword(s):

Mass Spectrometry ◽

Binding Sites ◽

Protein Complexes ◽

Type I ◽

Sds Page

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Nitrotyrosine Density of Rabbit Urinary Bladder Muscle and Mucosa Measured via Western Blotting and 96-Well Plate Analysis

ISRN Urology ◽

10.5402/2012/618247 ◽

2012 ◽

Vol 2012 ◽

pp. 1-5

Author(s):

Brittany Fitzpatrick ◽

Catherine Schuler ◽

Robert E. Leggett ◽

Robert M. Levin

Keyword(s):

Quantitative Analysis ◽

Western Blotting ◽

Optical Density ◽

Tissue Concentration ◽

Detrusor Muscle ◽

Pvdf Membrane ◽

Sds Page ◽

Wash Buffer ◽

Rabbit Bladder ◽

Plate Analysis

Purpose. Nitrotyrosine was quantitated in rabbit bladder muscle and mucosa using two analytical systems: Western blotting analyses and a 96-well plate quantitative analysis kit. Materials and Methods. Rabbit bladder muscle and mucosa were obtained from control rabbits. For the Western analysis, the samples were loaded into a SDS page gel and then transferred to a PVDF membrane. The optical density was measured using a Kodak Scanner. Using the 96-well plate, the samples and standards were loaded, incubated with primary and secondary antibody, washed and vacuumed with 10x wash buffer three times between each incubation period. Stop buffer was added to the plate and the results were quantified via the plate reader. Results. For both muscle and mucosa tissue, the optical density readings were linear with tissue concentration; the concentration of nitrotyrosine in the mucosa was significantly higher than in the muscle. However, whereas the Western blot analysis is based on relative optical densities, the 96-well plate kit provides a truly quantitative analysis. Discussion. Mucosa tissue displayed a higher density of nitrotyrosine than did detrusor muscle tissue. This may well be due to the significantly higher metabolic activity of the mucosa compared to the muscle.

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