Editing Isoelectric point (section)

== Peptides and proteins ==

A number of algorithms for estimating isoelectric points of [[peptide]]s and [[protein]]s have been developed. Most of them use [[Henderson–Hasselbalch equation]] with different pK values. For instance, within the model proposed by Bjellqvist and co-workers, the pKs were determined between closely related immobilines by focusing the same sample in overlapping pH gradients.<ref>{{Cite journal|last1=Bjellqvist|first1=B.|last2=Hughes|first2=G. J.|last3=Pasquali|first3=C.|last4=Paquet|first4=N.|last5=Ravier|first5=F.|last6=Sanchez|first6=J. C.|last7=Frutiger|first7=S.|last8=Hochstrasser|first8=D.|date=1993-10-01|title=The focusing positions of polypeptides in immobilized pH gradients can be predicted from their amino acid sequences|journal=Electrophoresis|volume=14|issue=10|pages=1023–1031|issn=0173-0835|pmid=8125050|doi=10.1002/elps.11501401163|s2cid=38041111}}</ref> Some improvements in the methodology (especially in the determination of the pK values for modified amino acids) have been also proposed.<ref>{{Cite journal|last1=Gauci|first1=Sharon|last2=van Breukelen|first2=Bas|last3=Lemeer|first3=Simone M.|last4=Krijgsveld|first4=Jeroen|last5=Heck|first5=Albert J. R.|date=2008-12-01|title=A versatile peptide pI calculator for phosphorylated and N-terminal acetylated peptides experimentally tested using peptide isoelectric focusing|journal=Proteomics|volume=8|issue=23–24|pages=4898–4906|doi=10.1002/pmic.200800295|issn=1615-9861|pmid=19003858|s2cid=21527631}}</ref><ref>{{Cite journal|last1=Gasteiger|first1=Elisabeth|last2=Gattiker|first2=Alexandre|last3=Hoogland|first3=Christine|last4=Ivanyi|first4=Ivan|last5=Appel|first5=Ron D.|last6=Bairoch|first6=Amos|date=2003-07-01|title=ExPASy: the proteomics server for in-depth protein knowledge and analysis|journal=Nucleic Acids Research|volume=31|issue=13|pages=3784–3788|issn=0305-1048|pmc=168970|pmid=12824418|doi=10.1093/nar/gkg563}}</ref> More advanced methods take into account the effect of adjacent amino acids ±3 residues away from a charged [[Aspartic acid|aspartic]] or [[glutamic acid]], the effects on free C terminus, as well as they apply a correction term to the corresponding pK values using [[genetic algorithm]].<ref>{{Cite journal|last1=Cargile|first1=Benjamin J.|last2=Sevinsky|first2=Joel R.|last3=Essader|first3=Amal S.|last4=Eu|first4=Jerry P.|last5=Stephenson|first5=James L.|date=2008-07-01|title=Calculation of the isoelectric point of tryptic peptides in the pH 3.5–4.5 range based on adjacent amino acid effects|journal=Electrophoresis|volume=29|issue=13|pages=2768–2778|doi=10.1002/elps.200700701|issn=0173-0835|pmid=18615785|doi-access=free}}</ref> Other recent approaches are based on a [[Support vector machine|support vector machine algorithm]]<ref>{{Cite journal|last1=Perez-Riverol|first1=Yasset|last2=Audain|first2=Enrique|last3=Millan|first3=Aleli|last4=Ramos|first4=Yassel|last5=Sanchez|first5=Aniel|last6=Vizcaíno|first6=Juan Antonio|last7=Wang|first7=Rui|last8=Müller|first8=Markus|last9=Machado|first9=Yoan J.|date=2012-04-03|title=Isoelectric point optimization using peptide descriptors and support vector machines|journal=Journal of Proteomics|volume=75|issue=7|pages=2269–2274|doi=10.1016/j.jprot.2012.01.029|issn=1876-7737|pmid=22326964}}</ref> and pKa optimization against experimentally known protein/peptide isoelectric points.<ref>{{Cite journal  | last1 = Kozlowski | first1 = LP. | title = IPC - Isoelectric Point Calculator. | journal = Biol Direct | volume = 11 | issue = 1 | pages = 55 | year = 2016 | doi = 10.1186/s13062-016-0159-9 | pmid = 27769290 | pmc=5075173 | doi-access = free }}</ref>

Moreover, experimentally measured isoelectric point of proteins were aggregated into the databases.<ref name="pmid15274128">{{Cite journal  | last1 = Hoogland | first1 = C. | last2 = Mostaguir | first2 = K. | last3 = Sanchez | first3 = JC. | last4 = Hochstrasser | first4 = DF. | last5 = Appel | first5 = RD. | title = SWISS-2DPAGE, ten years later. | journal = Proteomics | volume = 4 | issue = 8 | pages = 2352–6 | year = 2004 | doi = 10.1002/pmic.200300830 | pmid = 15274128 | s2cid = 31933242 }}</ref><ref name="pmid25252779">{{Cite journal  | last1 = Bunkute | first1 = E. | last2 = Cummins | first2 = C. | last3 = Crofts | first3 = FJ. | last4 = Bunce | first4 = G. | last5 = Nabney | first5 = IT. | last6 = Flower | first6 = DR. | title = PIP-DB: the Protein Isoelectric Point database. | journal = Bioinformatics | volume = 31 | issue = 2 | pages = 295–6 | year = 2015 | doi = 10.1093/bioinformatics/btu637 | pmid = 25252779 | doi-access = free }}</ref> Recently, a database of isoelectric points for all proteins predicted using most of the available methods had been also developed.<ref name="pmid27789699">{{Cite journal  | last1 = Kozlowski | first1 = LP. | title = Proteome-pI: proteome isoelectric point database. | journal = Nucleic Acids Res | year = 2016 | doi = 10.1093/nar/gkw978 | pmid = 27789699 | pmc=5210655 | volume=45| issue = D1 | pages = D1112–D1116 }}</ref>

In practice, a protein with an excess of basic aminoacids (arginine, lysine and/or histidine) will bear an isoelectric point roughly greater than 7 (basic), while a protein with an excess of acidic aminoacids (aspartic acid and/or glutamic acid) will often have an isoelectric point lower than 7 (acidic).
The electrophoretic linear (horizontal) separation of proteins by Ip along a pH gradient in a polyacrylamide gel (also known as [[isoelectric focusing]]), followed by a standard molecular weight linear (vertical) separation in a second polyacrylamide gel ([[SDS-PAGE]]), constitutes the so called [[two-dimensional gel electrophoresis]] or PAGE 2D. This technique allows a thorough separation of proteins as distinct "spots", with proteins of high molecular weight and low Ip migrating to the upper-left part of the bidimensional gel, while proteins with low molecular weight and high Ip locate to the bottom-right region of the same gel.