Existing users Log In New users Sign up

Isoelectric point of abnormal proteoforms in blood

DISCOVERIES (ISSN 2359–7232), 2016, October-December issue


Pirmoradian M, Aarsland D, Zubarev RA. Isoelectric point region pI≈7.4 as a treasure island of abnormal proteoforms in blood. Discoveries 2016, Jul-Sep; 4(4): e67 DOI: 10.15190/d.2016.14

Submitted: July 19th, 2016; Revised: November 16th, 2016; Accepted: November 18th, 2016; Published: December 1st, 2016;

 GO BACK to 2016, October-December issue


Isoelectric point region pI≈7.4 as a treasure island of abnormal proteoforms in blood

Mohammad Pirmoradian (1,2), Dag Aarsland (3), Roman A. Zubarev (1,*)

(1) Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden;

(2) Biomotif AB, Stockholm, Sweden;

(3) Alzheimer's Disease Research Centre, Department of Neurobiology, Care Sciences and Society, Karolinska Institutet, Stockholm, Sweden

*Correspondence toRoman A. Zubarev, PhD, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Scheeles väg 2, SE-17177 Stockholm, Sweden; Phone: +46 8 524 87594; E-mail: roman.zubarev@ki.se;


Theoretical distribution of isoelectric points (pI values) of human blood proteins exhibits multi-modality with a deep minimum in the range between pI 7.30 and 7.50. Considering that the pH of human blood is 7.4±0.1, normal forms of human proteins tend to eschew this specific pI region, thus avoiding charge neutrality that can result in enhanced precipitation. However, abnormal protein isoforms (proteoforms), which are the hallmarks and potential biomarkers of certain diseases, are likely to be found everywhere in the pI distribution, including this “forbidden” region. Therefore, we hypothesized that damaging proteoforms characteristic for neurodegenerative diseases are best detected around pI≈7.4. Blood serum samples from 14 Alzheimer's disease patients were isolated by capillary isoelectric focusing and analyzed by liquid chromatography hyphenated with tandem mass spectrometry. Consistent with the pI≈7.4 hypothesis, the 8 patients with fast memory decline had a significantly (p<0.003) higher concentration of proteoforms in the pI=7.4±0.1 region than the 6 patients with a slow memory decline. Moreover, protein compositions differed more from each other than for any other investigated pI region, providing absolute separation of the fast and slow decliner samples. The discovery of the “treasure island” of abnormal proteoforms in form of the pI≈7.4 region promises to boost biomarker development for a range of diseases.

Access full text of the manuscript here: 


1. Smith, L. M., Kelleher, N. L. & Consortium for Top Down, P. Proteoform: a single term describing protein complexity. Nat Methods, 2013; 10: 186-187. 

2. Tazi, J., Bakkour, N. & Stamm, S. Alternative splicing and disease. Biochim Biophys Acta, 2009; 1792: 14-26. 

3. Weiller, G. F., Caraux, G. & Sylvester, N. The modal distribution of protein isoelectric points reflects amino acid properties rather than sequence evolution. Proteomics, 2004; 4: 943-949. 

4. Theberge, R., Infusini, G., Tong, W., McComb, M. E. & Costello, C. E. Top-Down Analysis of Small Plasma Proteins Using an LTQ-Orbitrap. Potential for Mass Spectrometry-Based Clinical Assays for Transthyretin and Hemoglobin. Int J Mass Spectrom, 2011; 300: 130-142. 

5. Righetti, P. G., Sebastiano, R. & Citterio, A. Capillary electrophoresis and isoelectric focusing in peptide and protein analysis. Proteomics, 2013; 13: 325-340. 

6. Halligan, B. D. et al. ProMoST (Protein Modification Screening Tool): a web-based tool for mapping protein modifications on two-dimensional gels. Nucleic Acids Res, 2004; 32: W638-644.

7. Sillero, A. & Maldonado, A. Isoelectric point determination of proteins and other macromolecules: oscillating method. Comput Biol Med, 2006; 36: 157-166. 

8. Jang, B. G. et al. Plasma carbonic anhydrase II protein is elevated in Alzheimer's disease. J Alzheimers Dis, 2010; 21: 939-945.

9. Wu, S. et al. Multi-modality of pI distribution in whole proteome. Proteomics, 2006; 6: 449-455.

10. Chan, P., Lovric, J. & Warwicker, J. Subcellular pH and predicted pH-dependent features of proteins. Proteomics, 2006; 6: 3494-3501.

11. Chan, P. & Warwicker, J. Evidence for the adaptation of protein pH-dependence to subcellular pH. BMC Biol, 2009; 7: 69. 

12. Waugh A, G. A. Anatomy and Physiology in Health and Illness. 10th edition edn, (Churchill Livingstone Elsevier, 2007).

13. Halligan, B. D. ProMoST: a tool for calculating the pI and molecular mass of phosphorylated and modified proteins on two-dimensional gels. Methods Mol Biol, 2009; 527: 283-298. 

14. Chatterjee, R. et al. Isolation and characterization of a new hemoglobin derivative cross-linked between the alpha chains (lysine 99 alpha 1----lysine 99 alpha 2). J Biol Chem, 1986; 261: 9929-9937.

15. Carugo, O. Isoelectric points of multi-domain proteins. Bioinformation, 2007; 2, 101-104.

16. Pernemalm, M. & Lehtio, J. Mass spectrometry-based plasma proteomics: state of the art and future outlook. Expert review of proteomics, 2014; 11: 431-448.

17. Pirmoradian, M., Zhang, B., Chingin, K., Astorga-Wells, J. & Zubarev, R. A. Membrane-assisted isoelectric focusing device as a micropreparative fractionator for two-dimensional shotgun proteomics. Analytical chemistry, 2014; 86: 5728-5732.

18. Pirmoradian, M., Astorga-Wells, J. & Zubarev, R. A. Multijunction capillary isoelectric focusing device combined with online membrane-assisted buffer exchanger enables isoelectric point fractionation of intact human plasma proteins for biomarker discovery. Analytical chemistry, 2015.

19. Hye, A. et al. Plasma proteins predict conversion to dementia from prodromal disease. Alzheimers Dement, 2014; 10: 799-807, e792. 

20. Humpel, C. Identifying and validating biomarkers for Alzheimer's disease. Trends Biotechnol, 2011; 29: 26-32. 

21. Moya-Alvarado, G., Gershoni-Emek, N., Perlson, E. & Bronfman, F. C. Neurodegeneration and Alzheimer's disease (AD). What Can Proteomics Tell Us About the Alzheimer's Brain? Molecular & cellular proteomics : MCP, 2016; 15: 409-425. 

22. Lista, S., Faltraco, F., Prvulovic, D. & Hampel, H. Blood and plasma-based proteomic biomarker research in Alzheimer's disease. Prog Neurobiol, 2013; 101-102: 1-17.

23. Chingin, K., Astorga-Wells, J., Pirmoradian Najafabadi, M., Lavold, T. & Zubarev, R. A. Separation of polypeptides by isoelectric point focusing in electrospray-friendly solution using a multiple-junction capillary fractionator. Analytical chemistry, 2012; 84: 6856-6862.

24. Aarsland, D. et al. Frequency and case identification of dementia with Lewy bodies using the revised consensus criteria. Dement Geriatr Cogn Disord, 2008; 26: 445-452.

25. Doody, R. S., Massman, P. & Dunn, J. K. A method for estimating progression rates in Alzheimer disease. Arch Neurol, 2001; 58: 449-454.

26. Walker, J. M. The proteomics protocols handbook.  (Humana Press, 2005).

27. Altschul, S. F. A protein alignment scoring system sensitive at all evolutionary distances. J Mol Evol, 1993; 36, 290-300.

28. Krokhin, O. V. & Spicer, V. Predicting peptide retention times for proteomics. Curr Protoc Bioinformatics, 2010; 13: Unit 13 14. 

29. Pirmoradian, M. et al. Rapid and deep human proteome analysis by single-dimension shotgun proteomics. Molecular & cellular proteomics: MCP, 2013; 12: 3330-3338. 

30. Cox, J. & Mann, M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat Biotechnol, 2008; 26: 1367-1372.

31. Cox, J. et al. Accurate proteome-wide label-free quantification by delayed normalization and maximal peptide ratio extraction, termed MaxLFQ. Molecular & cellular proteomics: MCP, 2014; 13: 2513-2526.

32. Nanjappa, V. et al. Plasma Proteome Database as a resource for proteomics research: 2014 update. Nucleic Acids Res, 2014; 42: D959-965.

33. Cunningham, F. et al. Ensembl 2015. Nucleic Acids Res, 2015; 43: D662-669. 

34. Charif D., L. J. R. in Biological and Medical Physics, Biomedical Engineering   (ed Porto M. Bastolla U., Roman H.E., Vendruscolo M.)  207-232 (Springer Verlag, 2007).

35. Schjeide, B. M. et al. The role of clusterin, complement receptor 1, and phosphatidylinositol binding clathrin assembly protein in Alzheimer disease risk and cerebrospinal fluid biomarker levels. Arch Gen Psychiatry, 2011; 68: 207-213. 

36. Thambisetty, M. et al. Association of plasma clusterin concentration with severity, pathology, and progression in Alzheimer disease. Arch Gen Psychiatry, 2010; 67: 739-748. 

37. Kroksveen, A. C., Opsahl, J. A., Aye, T. T., Ulvik, R. J. & Berven, F. S. Proteomics of human cerebrospinal fluid: discovery and verification of biomarker candidates in neurodegenerative diseases using quantitative proteomics. J Proteomics, 2011; 74: 371-388. 

38. Zurbig, P. & Jahn, H. Use of proteomic methods in the analysis of human body fluids in Alzheimer research. Electrophoresis, 2012, 33: 3617-3630. 

39. de la Monte, S. M. Brain insulin resistance and deficiency as therapeutic targets in Alzheimer's disease. Curr Alzheimer Res, 2012; 9: 35-66.

40. Doecke, J. D. et al. Blood-based protein biomarkers for diagnosis of Alzheimer disease. Arch Neurol, 2012; 69: 1318-1325.

41. Bergamaschini, L. et al. Alzheimer's beta-amyloid peptides can activate the early components of complement classical pathway in a C1q-independent manner. Clin Exp Immunol, 1999; 115: 526-533.

42. Liao, P. C., Yu, L., Kuo, C. C., Lin, C. & Kuo, Y. M. Proteomics analysis of plasma for potential biomarkers in the diagnosis of Alzheimer's disease. Proteomics Clin Appl, 2007; 1: 506-512. 

43. Cross, A. J. et al. Reduced dopamine-beta-hydroxylase activity in Alzheimer's disease. Br Med J (Clin Res Ed), 1981; 282: 93-94.

44. Mustapic, M. et al. The catecholamine biosynthetic enzyme dopamine beta-hydroxylase (DBH): first genome-wide search positions trait-determining variants acting additively in the proximal promoter. Hum Mol Genet, 2014; 23: 6375-6384.

45. Bishnoi, R. J., Palmer, R. F., Royall, D. R. Vitamin D binding protein as a serum biomarker of Alzheimer's disease. J Alzheimers Dis, 2015; 43; 37-45.

46. Gressner, O. A. et al. Questioning the role of actinfree Gc-Globulin as actin scavenger in neurodegenerative central nervous system disease: relationship to S-100B levels and blood-brain barrier function. Clin Chim Acta, 2009; 400: 86-90.

47. Moon, M. et al. Vitamin D-binding protein interacts with Abeta and suppresses Abeta-mediated pathology. Cell Death Differ, 2013; 20: 630-638. 

48. Stein, T. D. & Johnson, J. A. Lack of neurodegeneration in transgenic mice overexpressing mutant amyloid precursor protein is associated with increased levels of transthyretin and the activation of cell survival pathways. J Neurosci, 2002; 22: 7380-7388.

49. Lazarov, O. et al. Environmental enrichment reduces Abeta levels and amyloid deposition in transgenic mice. Cell, 2005; 120: 701-713.

50. Comabella, M. et al. Cerebrospinal fluid chitinase 3-like 1 levels are associated with conversion to multiple sclerosis. Brain, 2010; 133: 1082-1093.

51. Yin, G. N., Lee, H. W., Cho, J. Y. & Suk, K. Neuronal pentraxin receptor in cerebrospinal fluid as a potential biomarker for neurodegenerative diseases. Brain Res, 2009; 1265: 158-170.

52. Hashimoto, Y. et al. Secreted calmodulin-like skin protein inhibits neuronal death in cell-based Alzheimer's disease models via the heterotrimeric Humanin receptor. Cell Death Dis, 2013; 4: e555

53. Petit-Turcotte, C. et al. Apolipoprotein C-I expression in the brain in Alzheimer's disease. Neurobiol Dis, 2001; 8: 953-963.

54. Plum, L., Schubert, M. & Bruning, J. C. The role of insulin receptor signaling in the brain. Trends Endocrinol Metab, 2005; 16: 59-65.

55. O'Day, D. H. & Myre, M. A. Calmodulin-binding domains in Alzheimer's disease proteins: extending the calcium hypothesis. Biochem Biophys Res Commun, 2004; 320: 1051-1054.

News & Events Latest news from Discoveries

  • March 2017 | PubMed inclusion News!

    Discoveries successfully passed the Scientific Quality Review by NLM-NIH for PubMedCentral and PubMed indexing (March 2017). This is the first and the most important step towards PubMedCentral and PubMed indexing! The second (last) step is the Technical Review, which should be faster and without problems.

  • April 2016 | Faster Peer-Review

    Starting on April 13th 2016, all articles selected for a peer-review will receive the post peer-review decision within 10 days. The initial pre-screening time will remain the same (48h from the submission of the manuscript). This decision will significantly accelerate the publication, with no effect on the quality of the peer-review process.

  • February 2016 | Manuscript submission

    Discoveries is commited to excellence, quality and high editorial standards. We are receiving an increasing number of manuscripts for which the identity of the authors/corresponding author can't be verified. Please NOTE that ALL these articles were and will be immediately REJECTED. Indicating an institutional email address is the easiest way to overcome this problem! Moreover, we do not accept any pressure on our editorial board to accept a manuscript. This results in a prompt rejection of the article.

    Editorial Policies
  • January 2016 | Main Objective

    After reaching all proposed milestones until now (including being indexed by Google Scholar in 2014), Discoveries' next Aim is PubMed indexing of all its articles (already published and upcoming). There will be no charge for the submission or publication of articles before Discoveries is indexed.

  • August 2015 | Discoveries - on PubMed

    We are happy to announce that our first Discoveries articles were included in PMC and PubMed. More articles (submitted by NIH funded authors) are now processed for being included.

    Discoveries articles now on PubMed
  • April 2015 | Special Issue

    DISCOVERIES published the SPECIAL ISSUE entitled "INFLAMMATION BETWEEN DEFENSE AND DISEASE: Impact on Tissue Repair and Chronic Sickness".

    Special Issue on "Inflammation"
  • 2015 | Ischemia Collection

    DISCOVERIES launched a call for papers for a Collection of Articles with focus on "ISCHEMIA". If you are interested to submit a manuscript, please contact us at info@discoveriesjournals.org

  • September 2014 | Special Issue

    DISCOVERIES just publish the SPECIAL ISSUE entitled "CELL SECRETION & MEMBRANE FUSION" in September 2014. Initially scheduled for publication between October 2014-March 2015, this issue was successfully published earlier than scheduled. 

    Special Issue
  • April 2014 | Indexed by Google Scholar

    All our published articles are now indexed by Google Scholar! First citations to Discoveries articles are included! Search for the article's title (recommended) or the authors:

    Google Scholar Search
  • 2014 | DISCOVERIES

    DOIs (Digital Object Identifiers) are now assigned to all our published manuscripts in Discoveries. DOI uniquely identifies an article and is provided by CrossRef.

  • July 2013 | Manuscript Submission

    Submit your manuscript FREE, FAST and EASY ! (in less than 1 minute)! There are NO fees for the manuscript submission or publishing of the accepted manuscripts.
    read more

  • July 2013 | DISCOVERIES

    We are now ACCEPTING MANUSCRIPTS for publishing in DISCOVERIES. We aim publishing a small number of high impact experimental articles & reviews (around 40/year) to maintain a high impact factor. Domains of interest: all areas related to Medicine, Biology and Chemistry ...

    read more
Member Login
Free Registration Click here to sign up
Copyright © 2013 Applied Systems. All Rights Reserved.