Isoelectric Point Of Tripeptide

[Escolastico Hall]

Theisoelectric point (pI) is the pH at which a molecule or a surface carries no net electrical charge. This means that the molecule or surface is electrically neutral, as the positive and negative charges present within it balance each other out. The concept of the isoelectric point is particularly important in biochemistry and chemistry, especially in the study of proteins, amino acids, and other biomolecules.

The isoelectric point (pI) stands as a critical concept in biochemistry, influencing various aspects of protein behavior, solubility, and pharmaceutical formulation. Its significance extends to protein purification techniques, such as chromatography and electrophoresis, where the manipulation of pH aids in separating proteins based on their charge differences. Moreover, the pI informs drug development strategies, guiding the formulation and optimization of pharmaceutical compounds for enhanced efficacy and stability. By comprehending the isoelectric point, researchers can navigate the complex interplay between pH, charge, and molecular structure, unlocking a multitude of applications across biotechnology, medicine, and chemistry.

Our peptide calculator is a convenient tool for scientists as a molecular weight peptide calculator, which can be used as an amino acid calculator as well. Additionally, the tool includes a hydrophobicity calculator, a net charge calculator at different pH, isoelectric point calculator and the hydrophilicity ratio.

To calculate the charge on a peptide you must add up the charges from all the positively charged components like the N-terminal amino group, arginine, lysine, and histidine residues, and then subtract the charges from all the negatively charged components like the C-terminal carboxyl group, aspartic acid, glutamic acid, cysteine, and tyrosine residues. The specific charge values depend on the pH and use dissociation constant (pKa) values for each ionizable group.

The peptide net charge calculator determines the charge of a peptide sequence at a given pH. It utilizes the Henderson-Hasselbalch equation and pKa values of the ionizable groups. The net charge (Z) sums the contributions from positive charges of the N-terminus, arginine, lysine, and histidine residues, and negative charges of the C-terminus, aspartic acid, glutamic acid, cysteine, and tyrosine residues.

The calculator is one of the most useful tool for the peptide chemist to calculate peptide molecular weight and more. With the calculator and its easy use, peptide chemists can have access to a molecular weight peptide calculator and amino acid calculator, the isoelectric point, a peptide net charge calculator at neutral pH, the average hydrophilicity, the percentage of hydrophilic amino acids, the plot of the net charge vs. pH and a hydrophobicity calculator displayed in a plot.

For the molecular weight amino acid calculator, you can enter the 1- or 3- letter code of the desired amino acid, and the tool will provide the value the same way it would calculate peptide molecular weight.

The calculation of the average hydrophilicity of a peptide is based on the data from Hopp&Woods. The hydrophilicity value for each amino acid in the peptide sequence is indicated in a bar graph. The ratio of hydrophilic residues to total number of amino acids is reported in %.

To use our peptide calculator mass properties, enter the sequence or the amino acid using 1-letter or 3-letter amino acid codes and our calculator will provide the following physico-chemical properties of the sequence:

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Profiling of phosphorylation changes on a large scale (phosphoproteomics) can be performed by mass-spectrometry (MS). However, identification of phosphorylated peptides is challenging due to their low abundance and poor ionization efficiency4, thus methodological improvements are needed to increase the analytical depth. Current methods include reduction of sample complexity both by extensive sample fractionation and by specific phospho-peptide enrichment strategies5. These approaches most often demand long MS-analysis time and high amount of starting material, hence influencing throughput and cost of the analysis, and may not even be feasible in cases where the availability of starting material is limited. Labeling strategies allow parallel quantitative analysis of multiple samples (multiplexing), therefore reducing the analysis time as compared with sequential sample analysis performed with label-free approaches. Furthermore, multiplexing also results in reduced quantitative variability6, 7. Metabolic labeling methods, especially SILAC, have been broadly employed for phosphoproteomics analysis5, but their multiplexing capability is limited to two or three samples. Isobaric labels, such as Tandem Mass Tags (TMT), allow simultaneous quantification of up to ten samples. A limited number of phosphoproteomics studies utilizing isobaric labeling have been reported so far, identifying in between 11,000 to 26,000 phosphorylation sites8,9,10,11,12,13, however they require high amount of starting material or labeling reagents.

Previously, we described a method for precise fractionation of peptides based on isoelectric point (high-resolution isoelectric focusing, HiRIEF). Furthermore, we demonstrated that when coupled to liquid chromatography and mass spectrometry (HiRIEF LC-MS), the method enables high coverage of the cellular proteome14. In the current study, we explore how HiRIEF LC-MS performs in a quantitative phosphoproteomics workflow. Samples from HeLa cells were enriched for phosphorylated peptides employing titanium dioxide (TiO2), followed by isobaric labeling and HiRIEF LC-MS. Performance of the developed workflow resulted in the identification of 22,712 phosphorylation sites across 10 samples, of which 1,264 were not previously reported in the PhosphoSitePlus database15. Finally, functionality of such novel phosphorylation sites was predicted to indicate sites with putative biological functions.

The HiRIEF pre-fractionation method enables in-depth proteome analysis by highly reducing the peptide complexity in the fractions analyzed by LC-MS14. In order to investigate how this method performs in a quantitative phosphoproteomics setting, we combined it with titanium dioxide based phospho-peptide enrichment and isobaric labeling (Fig. 1). Isobaric labeling of peptides using TMT 10-plex reagents allows for relative quantification of up to ten different samples in a single MS experiment. Control cells were harvested in exponential growth phase (four biological replicates) to represent the phosphoproteome in an asynchronous cell population (Supplementary Fig. S1b). Experimental conditions included pervanadate treatment and mitotic arrest (three biological replicates each) and were selected to enrich for different phosphorylation events. Pervanadate inhibits protein tyrosine phosphatases16, 17, resulting in dramatically increased levels of tyrosine phosphorylation as assayed by western blot (Supplementary Fig. S1a), without affecting the cell cycle distribution (Supplementary Fig. S1b). Mitotic arrest achieved by double thymidine block and nocodazole treatment induces extensive serine and threonine phosphorylation18. Flow cytometric cell cycle analysis confirmed the efficiency of the induction of mitotic arrest (Supplementary Fig. S1b). The human cervix adenocarcinoma cell line HeLa was used as a model system as it is a well characterized model for phosphorylation studies, especially in relation to mitosis18,19,20.

Finally, a subset of 2,050 phospho-peptides commonly identified in both IPG strips (Fig. 2c) is attributed to the overlap in their pH ranges (Supplementary Fig. S3), demonstrating non-redundant fractionation of peptides based on their isoelectric point.

Functional characterization of the identified phospho-sites. (a) Heatmap representing complete linkage hierarchical clustering based on Euclidian distance of the unique phospho-site ratios. Row color-coding represents the modified residue (S,T or Y). (b) Venn diagram showing the overlap between the identified phospho-sites, the ones previously reported in the PhosphoSitePlus database and the HeLa phospho-sites reported by Sharma et al. in 2014. (c) Proportion of novel and known phospho-sites per modified residue. (d) Distribution of protein precursor areas for the novel and known phospho-sites per type of modified residue. Two-sided t-test was performed to assess significance.

To define phospho-sites significantly regulated upon pervanadate treatment or mitotic arrest we used a Benjamini-Hochberg corrected t-test p-value of less than 0.01 and a fold change of more than two when comparing treated to control cells. After pervanadate treatment and cell cycle arrest 3,254 and 11,031 phospho-sites were found significantly regulated respectively (Supplementary Fig. S6). To examine whether phosphorylation sites are differentially regulated when identified in singly or in multiply phosphorylated peptides, we examined the subset of 1,810 phospho-sites that were commonly identified in peptides carrying single or multiple phosphorylations. Upon mitotic arrest, 130 of those phospho-sites are significantly regulated when identified in singly phosphorylated peptides but are not regulated when identified in multiply phosphorylated peptides. Conversely, 613 phospho-sites are significantly regulated only when identified in multiply phosphorylated peptides but not in singly phosphorylated peptides (Supplementary Fig. S7). Such regulation events that are detected exclusively in the context of multisite phosphorylation would not be apparent when analyzing only singly phosphorylated peptides, thus illustrating the value of detecting multiply phosphorylated peptides.

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