Protein Identification by Mass Spectrometry: Proteomics

  • Melinda Wojtkiewicz
  • Kelley Barnett
  • Pawel CiborowskiEmail author
Part of the Springer Protocols Handbooks book series (SPH)


Identification, characterization, and quantitation of changes of proteins induced by various types of external and/or internal stimuli are the major objectives of proteomics. Because proteins consist of 20 amino acids, their sequencing is more complicated than nucleic acids, which are a combination of four nucleotides. Proteins and peptides cannot be amplified and/or hybridized in vitro as nucleic acids. Moreover, proteins may have multiple posttranslationally modified sites and multiple types of modifications, such as phosphorylation, nitration, acetylation, and methylation, just to mention a few. These facts have a decisive impact on analytical methods used for protein sequencing and characterization. Before the development of soft ionization (the formation of ions without breaking any chemical bonds) techniques, proteins and peptides were sequenced using the Edman degradation method, based on the sequential removal of N-terminal amino acids (Edman 1970). This method, although still used (Mari et al. 2010), has many limitations such as an inability to sequence peptides that have the N-terminus blocked (Chen et al. 2008). Fragmentation of relatively short peptides (usually 6–30 amino acids in length) in gas phase in mass spectrometers exponentially increased the confidence with which we are able to determine peptide sequence and reveal other structural features, including posttranslational modifications (Biemann 1988; Strachunskii et al. 1992; Appella et al. 2000; Meng et al. 2005; Paizs and Suhai 2005). A number of specific proteolytic enzymes are available to fragment larger proteins into shorter peptides more suitable for high-quality analysis in tandem mass spectrometry (MS/MS) (see Fig. 28.3). The most commonly used enzyme, trypsin, specifically cleaves the peptide bond C-terminal to arginine and lysine residues with any amino acid excluding proline (Thiede et al. 2005). The Arg/Lys–Pro peptide bond is cleaved at a very low rate; however, if such a peptide is well ionized, it still can be detected and sequenced by mass spectrometry (Pottiez et al. 2010)


Neural stem cell Neural progenitor cells Neurosphere Differentiation 


  1. Ahmed FE (2008) Utility of mass spectrometry for proteome analysis: part I. Conceptual and experimental approaches. Expert Rev Proteomics 5:841–864CrossRefPubMedGoogle Scholar
  2. Ahmed FE (2009) Sample preparation and fractionation for proteome analysis and cancer biomarker discovery by mass spectrometry. J Sep Sci 32:771–798CrossRefPubMedGoogle Scholar
  3. Appella E, Arnott D, Sakaguchi K, Wirth PJ (2000) Proteome mapping by two-dimensional polyacrylamide gel electrophoresis in combination with mass spectrometric protein sequence analysis. EXS 88:1–27PubMedGoogle Scholar
  4. Biemann K (1988) Contributions of mass spectrometry to peptide and protein structure. Biomed Environ Mass Spectrom 16:99–111CrossRefPubMedGoogle Scholar
  5. Bodzon-Kulakowska A, Bierczynska-Krzysik A, Dylag T, Drabik A, Suder P, Noga M, Jarzebinska J, Silberring J (2007) Methods for samples preparation in proteomic research. J Chromatogr B Analyt Technol Biomed Life Sci 849:1–31CrossRefPubMedGoogle Scholar
  6. Canas B, Pineiro C, Calvo E, Lopez-Ferrer D, Gallardo JM (2007) Trends in sample preparation for classical and second generation proteomics. J Chromatogr A 1153:235–258CrossRefPubMedGoogle Scholar
  7. Chen W, Yin X, Yin Y (2008) Rapid and reliable peptide de novo sequencing facilitated by microfluidic chip-based Edman degradation. J Proteome Res 7:766–770CrossRefPubMedGoogle Scholar
  8. Domon B, Aebersold R (2006) Mass spectrometry and protein analysis. Science 312:212–217CrossRefPubMedGoogle Scholar
  9. Edman P (1970) Sequence determination. Mol Biol Biochem Biophys 8:211–255PubMedGoogle Scholar
  10. Granvogl B, Ploscher M, Eichacker LA (2007) Sample preparation by in-gel digestion for mass spectrometry-based proteomics. Anal Bioanal Chem 389:991–1002CrossRefPubMedGoogle Scholar
  11. Grimm RL, Beauchamp JL (2002) Evaporation and discharge dynamics of highly charged droplets of heptane, octane, and p-xylene generated by electrospray ionization. Anal Chem 74:6291–6297CrossRefPubMedGoogle Scholar
  12. Gundry RL, White MY, Murray CI, Kane LA, Fu Q, Stanley BA, Van Eyk JE (2009) Preparation of proteins and peptides for mass spectrometry analysis in a bottom-up proteomics workflow. Curr Protoc Mol Biol Chapter 10:Unit10.25Google Scholar
  13. Li KY, Tu H, Ray AK (2005) Charge limits on droplets during evaporation. Langmuir 21:3786–3794CrossRefPubMedGoogle Scholar
  14. Luque-Garcia JL, Neubert TA (2007) Sample preparation for serum/plasma profiling and biomarker identification by mass spectrometry. J Chromatogr A 1153:259–276CrossRefPubMedGoogle Scholar
  15. Mari A, Ciardiello MA, Tamburrini M, Rasi C, Palazzo P (2010) Proteomic analysis in the identification of allergenic molecules. Expert Rev Proteomics 7:723–734CrossRefPubMedGoogle Scholar
  16. McGregor E, De Souza A (2006) Proteomics and laser microdissection. Methods Mol Biol 333:291–304PubMedGoogle Scholar
  17. Meng F, Forbes AJ, Miller LM, Kelleher NL (2005) Detection and localization of protein modifications by high resolution tandem mass spectrometry. Mass Spectrom Rev 24:126–134CrossRefPubMedGoogle Scholar
  18. Paizs B, Suhai S (2005) Fragmentation pathways of protonated peptides. Mass Spectrom Rev 24:508–548CrossRefPubMedGoogle Scholar
  19. Pottiez G, Haverland N, Ciborowski P (2010) Mass spectrometric characterization of gelsolin isoforms. Rapid Commun Mass Spectrom 24:2620–2624CrossRefPubMedPubMedCentralGoogle Scholar
  20. Roepstorff P (2000) MALDI-TOF mass spectrometry in protein chemistry. EXS 88:81–97PubMedGoogle Scholar
  21. Rozek W, Ricardo-Dukelow M, Holloway S, Gendelman HE, Wojna V, Melendez L, Ciborowski P (2007) Cerebrospinal fluid proteomic profiling of HIV-1-infected patients with cognitive impairment. J Proteome Res 6:4189–4199CrossRefPubMedGoogle Scholar
  22. Rozek W, Horning J, Anderson J, Ciborowski P (2008) Sera proteomic biomarker profiling in HIV-1 infected subjects with cognitive impairment. Proteomics Clin Appl 2:1498–1507CrossRefPubMedPubMedCentralGoogle Scholar
  23. Seidler J, Zinn N, Boehm ME, Lehmann WD (2010) De novo sequencing of peptides by MS/MS. Proteomics 10:634–649CrossRefPubMedGoogle Scholar
  24. Silberring J, Ciborowski P (2010) Biomarker discovery and clinical proteomics. Trends Analyt Chem 29:128CrossRefPubMedPubMedCentralGoogle Scholar
  25. Strachunskii LS, Prokhorenkov PI, Novikova Iu A, Krechikova OI, Iakusheva LV (1992) [Use of cefoperazone in clinical practice]. Antibiot Khimioter 37:19–21PubMedGoogle Scholar
  26. Thiede B, Hohenwarter W, Krah A, Mattow J, Schmid M, Schmidt F, Jungblut PR (2005) Peptide mass fingerprinting. Methods 35:237–247CrossRefPubMedGoogle Scholar
  27. Wilm M (2011) Principles of electrospray ionization. Mol Cell Proteomics 10(M111):009407PubMedGoogle Scholar
  28. Yates JR 3rd (1998) Mass spectrometry and the age of the proteome. J Mass Spectrom 33:1–19CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Melinda Wojtkiewicz
    • 1
  • Kelley Barnett
    • 1
  • Pawel Ciborowski
    • 1
    Email author
  1. 1.Department of Pharmacology and Experimental NeuroscienceUniversity of Nebraska College of MedicineOmahaUSA

Personalised recommendations