Stabilization of Proteins and Noncovalent Protein Complexes during Electrospray Ionization by Amino Acid Additives

2015 ◽  
Vol 87 (14) ◽  
pp. 7433-7438 ◽  
Author(s):  
Hua Zhang ◽  
Haiyan Lu ◽  
Konstantin Chingin ◽  
Huanwen Chen
Keyword(s):  
Amino Acid ◽  
Electrospray Ionization ◽  
Protein Complexes ◽  
Noncovalent Protein

Gas Phase Stabilization of Noncovalent Protein Complexes Formed by Electrospray Ionization

2009 ◽  
Vol 81 (18) ◽  
pp. 7801-7806 ◽  
Author(s):  
Dhanashri Bagal ◽  
Elena N. Kitova ◽  
Lan Liu ◽  
Amr El-Hawiet ◽  
Paul D. Schnier ◽  
...  
Keyword(s):  
Electrospray Ionization ◽  
Gas Phase ◽  
Protein Complexes ◽  
Phase Stabilization ◽  
Noncovalent Protein

Mass Spectrometry for Proteomics and Recent Developments in ESI, MALDI and other Ionization Methodologies

2019 ◽  
Vol 16 (4) ◽  
pp. 267-276
Author(s):  
Qurat ul Ain Farooq ◽  
Noor ul Haq ◽  
Abdul Aziz ◽  
Sara Aimen ◽  
Muhammad Inam ul Haq
Keyword(s):  
Mass Spectrometry ◽  
Electrospray Ionization ◽  
New Technologies ◽  
Protein Complexes ◽  
Imaging Techniques ◽  
Desorption Electrospray Ionization ◽  
High Throughput Analysis ◽  
Enhanced Sensitivity ◽  
Recent Developments ◽  
The Way

Background: Mass spectrometry is a tool used in analytical chemistry to identify components in a chemical compound and it is of tremendous importance in the field of biology for high throughput analysis of biomolecules, among which protein is of great interest. Objective: Advancement in proteomics based on mass spectrometry has led the way to quantify multiple protein complexes, and proteins interactions with DNA/RNA or other chemical compounds which is a breakthrough in the field of bioinformatics. Methods: Many new technologies have been introduced in electrospray ionization (ESI) and Matrixassisted Laser Desorption/Ionization (MALDI) techniques which have enhanced sensitivity, resolution and many other key features for the characterization of proteins. Results: The advent of ambient mass spectrometry and its different versions like Desorption Electrospray Ionization (DESI), DART and ELDI has brought a huge revolution in proteomics research. Different imaging techniques are also introduced in MS to map proteins and other significant biomolecules. These drastic developments have paved the way to analyze large proteins of >200kDa easily. Conclusion: Here, we discuss the recent advancement in mass spectrometry, which is of great importance and it could lead us to further deep analysis of the molecules from different perspectives and further advancement in these techniques will enable us to find better ways for prediction of molecules and their behavioral properties.

scholarly journals Pichia pastoris and the Recombinant Human Heterodimeric Amino Acid Transporter 4F2hc-LAT1: From Clone Selection to Pure Protein

2021 ◽  
Vol 4 (3) ◽  
pp. 51
Author(s):  
Satish Kantipudi ◽  
Daniel Harder ◽  
Sara Bonetti ◽  
Dimitrios Fotiadis ◽  
Jean-Marc Jeckelmann
Keyword(s):  
Amino Acid ◽  
Pichia Pastoris ◽  
Protein Complexes ◽  
Amino Acid Transporter ◽  
Amino Acid Transporters ◽  
Expression Host ◽  
Amino Acids And Derivatives ◽  
Heterodimeric Complex ◽  
Acid Transporter ◽  
And Function

Heterodimeric amino acid transporters (HATs) are protein complexes composed of two subunits, a heavy and a light subunit belonging to the solute carrier (SLC) families SLC3 and SLC7. HATs transport amino acids and derivatives thereof across the plasma membrane. The human HAT 4F2hc-LAT1 is composed of the type-II membrane N-glycoprotein 4F2hc (SLC3A2) and the L-type amino acid transporter LAT1 (SLC7A5). 4F2hc-LAT1 is medically relevant, and its dysfunction and overexpression are associated with autism and tumor progression. Here, we provide a general applicable protocol on how to screen for the best membrane transport protein-expressing clone in terms of protein amount and function using Pichia pastoris as expression host. Furthermore, we describe an overexpression and purification procedure for the production of the HAT 4F2hc-LAT1. The isolated heterodimeric complex is pure, correctly assembled, stable, binds the substrate L-leucine, and is thus properly folded. Therefore, this Pichia pastoris-derived recombinant human 4F2hc-LAT1 sample can be used for downstream biochemical and biophysical characterizations.

scholarly journals Multiple components of arginine and phenylalanine transport induced in neutral and basic amino acid transporter-cRNA-injected Xenopus oocytes

1996 ◽  
Vol 318 (3) ◽  
pp. 915-922 ◽  
Author(s):  
George J PETER ◽  
Iain G. DAVIDSON ◽  
Aamir AHMED ◽  
Lynn McILROY ◽  
Alexander R. FORRESTER ◽  
...  
Keyword(s):  
Amino Acid ◽  
Xenopus Oocytes ◽  
Competitive Inhibition ◽  
Protein Complexes ◽  
Basic Amino Acid ◽  
Amino Acid Transporter ◽  
Transport Processes ◽  
Arginine Transport ◽  
Acid Transporter ◽  
Phenylalanine Transport

The induced uptakes of l-[3H]phenylalanine and l-[3H]arginine in oocytes injected with clonal NBAT (neutral and basic amino acid transporter) cRNA show differential inactivation by pre-treatment with N-ethylmaleimide (NEM), revealing at least two distinct transport processes. NEM-resistant arginine transport is inhibited by leucine and phenylalanine but not by alanine or valine; mutual competitive inhibition of NEM-resistant uptake of arginine and phenylalanine indicates that the two amino acids share a single transporter. NEM-senstive arginine transport is inhibited by leucine, phenylalanine, alanine and valine. At least two NEM-sensitive transporters may be expressed because we have been unable to confirm mutual competitive inhibition between arginine and phenylalanine transport. The NEM-resistant transport mechanism appears to involve distinct but overlapping binding sites for cationic and zwitterionic substrates. NBAT is known to form oligomeric protein complexes in cell membranes, and its functional roles when expressed in Xenopus oocytes may include interaction with oocyte proteins, leading to increased native amino acid transport activities; these resemble NBAT-expressed activities in terms of NEM-sensitivity and apparent substrate range (including an unusual inhibition by β-phenylalanine).

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