Mass Spectrometry Essay

This experiment was conducted as per the BCHM 310 Laboratory Manual [3]. The first objective of this experiment was to analyze the purity of the invertase fractions collected during experiment 6, and to determine the molecular weight of LDH-H4, LDH-M4 and invertase subunits. This was accomplished using sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS-PAGE). In this procedure, SDS, a negatively charged amphipathic molecule, was used to denature the proteins and to give each protein a similar charge-to-mass ratio [4]. As a result, most oligomeric proteins separated into individual subunits, and each subunit assumed a rod-like shape [4]. The distance travelled by each subunit, along the polyacrylamide gel, was a function of its molecular weight; where proteins with a greater molecular weight moved a smaller distance than proteins with lower weights [5]. Since SDS is not a reducing agent, and no other reducing agent was added, oligomers with disulfide bonds between subunits would have remained intact [4]. However, this was not expected to be problematic for analyzing invertase or LDH isozymes, as these proteins lack disulfide interactions between their subunits [2,6]. In addition, since invertase and LDH are homo-oligomers, each protein’s subunits were expected to migrate the same distance [2,6].

This technique separates Rubisco samples based on their size. The electrophoresis has a positive and a negative end. Positive charge proteins are loaded from the positive end and migrate towards the negative end. Negative charge proteins are loaded from the negative end and migrate towards the positive end (Sakthivel & Palani, 2016). The sample that contained the highest molecular weight of Rubisco will travel the shortest distance on the gel while the protein with the smallest molecular weight will travel the longest distance (Sakthivel & Palani, 2016). The size proportion of each Rubisco molecule correlates with the distance traveled. Rubisco will be in its purest form after running through SDS-page since each technique will increase the purity of the protein. If the salting out, the ion exchange and the SDS-page protein isolation techniques are performed on protein Rubisco, then it is purified and separated by solubility, charge, and size. The rationale of this experiment is to isolate the purest form of Rubisco so that it can perform carbon fixation at an optimal

In UNIT 7 you learned about the basics structure as well as the basic functions of deoxyribonucleic acid. You learned that deoxyribonucleic acid carries an “instruction manuel” to produce proteins that are responsible for passing traits from your parents to you. As you demonstrated in the previous activity, deoxyribonucleic acid is made of four nucleotides base pairs: adenine, thymine, cytosine, and guanine. They are often abbreviated as A, T, C, and G. The uniqueness of deoxyribonucleic acid is not particularly in just these 4 subunits, but how they are arranged. In this activity you will recreate protein synthesis.

This assignment will outline the function of proteins in living organisms and the important roles of different types of protein. “Protein composes 10-30% of cell mass and is the basic structural material of the body” (Marieb E.N.M et al, 2004). Protein is a nutrient that living organisms need to exist and grow, as well as water being a key feature. “All protein contains carbon, oxygen, hydrogen and nitrogen” (Marieb E.N.M et al, 2004). Amino acids form links of 20, “The sequences at which they are bound together produces proteins that vary widely in both structure and function” (Marieb E.N.M et al, 2004).

The testing of various proteins was performed by comparing the molecular weight of proteins using SDS PAGE. The molecular focus in the lab was the testing of proteins, which are macromolecules consisting of amino acid monomers linked through chemical bonds. These proteins have a hierarchy of structure that consists of folding that determines the direct function of each protein.. The molecular weight of these proteins were measured using SDS PAGE. SDS PAGE stands for sodium dodecylsulfate polyacrylamide gel electrophoresis. SDS is an anion detergent that binds with the protein structures and causes them to separate due to the change in bonding charge. SDS and heat are how the proteins are denatured. The process of denaturing a protein is breaking

In 1988, Fenn had published two articles that made a large development for mass spectrometry with “electrospray” for macromolecules. In the first article, his studies of the polyethylene glycol molecules of an unknown mass demonstrated that the method could handle large molecule masses of high charges, while the second one showed the use of the method with medium-sized whole protein also. The release of ions is produced by spraying the sample with an electrical field so that charged droplets are produced. Ultimately, the water will evaporate and “stark naked” protein molecules stays. This

Introduction: The goal of this experiment was to practice using the FirstGlance in Jmol molecular visualization to examine key structural features of proteins. This work is important because protein structure can be related to function, multiple-sequence alignments and evolutionary preservation, and designing drug. FirstGlance in Jmol makes it fairly easy to perceive structure-function relationships in the protein you chose. Using FirstGlance, it is easy to visualize and distinguish chains, and disulfide bonds are obvious. Alpha helices and beta strands are evident due to the "cartoon" secondary structural schematic.

Our goal in this experiment is to determine the reaction rate and specific activity of the protein extract. Also, the purpose of this lab is to characterize Vmax and Km for catechol and L-Dopa. Along with, determining whether the mode of inhibition by PTU is competitive or non-competitive? We performed a spectrometry analysis in order to determine the reaction rate of PPO, specific activity, and substrate- enzyme relationships among L-Dopa, catechol, and PTU. The reaction rate of polyphenol oxidase determined to be 0.04932957 μmin/min. By incoporating Beer’s Law, the specific activity of our protein extract was 0.2517127 μm/mgmin.In order to determine Vmax and Km for catechol and L-dopa we had to take averages from 3 dfferent groups. The Vmax for L-dopa determined to be 14.27, the ½ Vmax was 7.134, and the Km value was 0.485. The Vmax for catechol determined to be 10.00, ½ Vmax was 5.33, and the Km value was 0.38. The mode of inhibition by PTU determined to be non- competitive. The graph shows a rough hyperbolic trend with the slop increasing and then slowly making a curve towards the end for the catechol substrate concentration. However, the reaction with catechol and PTU portrayes a flat line. This is due to the fact that the increasing amount of substrate concentration had no effect on the reaction at all. Therefore, PTU was inhibiting the

Macromolecules rely on their environment for proper folding of their respective three- dimensional structure.10 This makes intuitive sense since the exterior, which is polar, is making contact to a polar solvent, like the cytoplasm that provide hydrogen bonds, electrostatic interactions, and conceal the nonpolar residues away from the polar solvent.10 The body has a stable pH and temperature that proteins take advantage to form amazing functional complexes. They can catalyze reactions as enzymes, transport compounds, and make complex units to reach a certain goal like ATP synthase10.They can be dangerous as well, such as binding to receptors on cells for virus entry4. Proteins lose their conformation when they are out of range in temperature

Protein purification is a process that can be employed to separate a single protein from a larger starting material which may be anything from an organ to a cell. Isolating a purified protein from a larger fraction enables further analysis such as determination of amino acid sequence, potential biological function, and even evolutionary relationship. (Cuatrecasas 1970) In this experiment, the enzyme lactate dehydrogenase will be purified, this enzyme is found extensively in human cells and catalyzes the conversion of lactate to pyruvate, an essential part in energy production. LDH is a key part of anaerobic energy production especially within glycolysis in which LDH catalyzes the conversion of the reverse reaction, pyruvate to lactate, generating NAD+ from NADH, reproducing the oxidized form of the coenzyme which can be used for oxidative respiration. (Markert 1963) Due to the fact that number of purification steps correlates with the purity of the protein multiple purification techniques will be used to isolate a pure form of LDH. LDH will be isolated from a larger “cytosol” fraction collected from a homogenized rat liver in a previous fractionation exercise. Of the procedures that will be used to isolate and purify proteins from a larger fractionate are a set of techniques collectively known as chromatography. These techniques all have the same premise, in that they consist of a stationary phase, also known as the

350 of each sample are loaded on the gel. Based on Genomic Solutions, once fixing the changes of fresh 40% methanol and 10% glacial acetic acid for every 12 hour each, the gels were stained for 12 hours with Sypro Ruby solution in the dark. The gels were incubated in 10% methanol: 6% acetic acid for 4 hours for destaining procedure. The gels are the imaged by using ProPick Workstation. Triangulation and robotic excision are conducted for identification of protein spots. It was then submitted to tandem mass spectrometric analysis.

Proteins are one of the main macromolecules that are important for human function. This study examines the presence of proteins in the given samples. The Biuret reagent created a color transitioned on the various samples. The Biuret reagent is an aqueous solution that is composed of sodium hydroxide and copper (II) sulfate. The copper ions in the Biuret reagent react with the peptide bonds which cause the color change in the test samples. The amount of protein in the various samples determined the color transition. The sample solutions with lower presence of protein did not change color as opposed to the sample solutions with a higher presence of

GST- and MBP- tagged proteins were overexpressed at the similar levels, however, protein recovery in a 100.000g supernatant (soluble fraction) was very poor (data not shown). Additionally GST-tagged proteins were irreversibly sticking to the column which made us focus exclusively on the MBP-tag proteins. Temperature variations (160C – 370C), IPTG concentration (0.5mM – 1mM), duration of induction (3h – 18h), as well as the addition of various molecules (arginine, DTT, glycine) to the supernatant, were tested in a number of experiments to optimize the expression and recovery of recombinant proteins (data not shown). In short, protein expression at 160C with an induction with 0.5mMIPTG for 18h was chosen as optimal. Addition of 1M arginine to the homogenate was also shown to be beneficial for protein recovery, especially after freeze/though cycles. In Figure 1A a representative example of one such an experiment is shown.

The very first purification step involved the 65% ammonium sulfate cut that resulted in a highest protein concentration out of all the purification steps. The protein concentration went up from 3.925mg/ml from the clarified homogenate to 28.11mg/ml, which is resulted of the pelleting out of the LDH and reducing the volume from 114mL to 5.7mL. Furthermore, the 65% pellet cut was able to recovery 46% of the LDH, while purifying it by 6.8 fold. Also, this step was able to remove 65% of the total proteins, while retaining 35% of it. Due to the increased purity and 46% recovery of LDH we can conclude that most of the removed total protein was non-LDH, contaminating protiens. Unfortunately, since this was only the first purification step, the amount of comtaminating proteins is still fairly high as depicted in Figure 14, where the 65% cut pellet contains several dark bands besides the one at 35kDa.

Now, homogenization for sample preparation in proteomics can be done by the following 5 ways: -