Summary
Ladder sequencing of polypeptides involves progressive N- or C-terminal amino acid truncation via chemical or enzymatic treatments. Peptide ladders are generated in which each component differs from the next by one residue. The ladder components are analyzed by mass spectrometry, and the amino acid sequence is deduced from the mass differences between consecutive fragments. Chemical procedures are common in N-terminal degradation, whereas proteolytic digestion is often used in C-terminal sequence analysis. Matrix-assisted laser desorption/ionization mass spectrometry is widespread for one-step readout of the peptide ladders and provides high sensitivity in combination with robustness and ease of use. The particular advantage of ladder sequencing in relation to other techniques for sequence analysis is the high data acquisition rate and the very good sample throughput that can be achieved. Multiple determinations are carried out within minutes at high sensitivity and low sample consumption. Several reports demonstrate analysis at the low picomole to femtomole level.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Preview
Unable to display preview. Download preview PDF.
References
Edman P (1950) Method for determination of the amino acid sequence in peptides. Acta Chem Scand 4: 283–293
Chait BT, Wang R, Beavis RC, Kent SBH (1993) Protein ladder sequencing. Science 262: 89–92
Bartlet-Jones M, Jeffery WA, Hansen HF, Pappin, DJC (1994) Peptide ladder sequencing by mass spectrometry using a novel, volatile degradation reagent. Rapid Commun Mass Spectrom 8: 737–742
Godovac-Zimmermann J, Krause I, Baranyi M, Fischer-Fruhholz S, Juszczak J, Erhardt G, Buchberger J, Klostermeyer H (1996) Isolation and rapid sequence characterization of two novel bovine p-lactoglobulins I and J. J Prot Chem 15: 743–750
Gu Q-M, Prestwich GD (1997) Efficient peptide ladder sequencing by MALDI-TOF mass spectrometry using allyl isothiocyanate. J Peptide Res 49: 484–491
Tsugita A, Takamoto K, Kamo M, Iwadate H (1992) C-terminal sequencing of protein. A novel partial acid hydrolysis and analysis by mass spectrometry. Eur J Biochem 206: 691–696
Takamoto K, Kamo M, Kubota K, Satake K, Tsugita A (1995) Carboxy-terminal degradation of peptides using perfluoroacyl anhydrides. A C-terminal sequencing method. Eur J Biochem 228: 362–372
Hayashi R, Moore S, Stein WH (1973) Carboxypeptidase from yeast. Large scale preparation and the application to COOH-terminal analysis of peptides and proteins. J Biol Chem 248: 2296–2302
Klarskov K, Breddam K, Roepstorff P (1989) C-terminal sequence determination of peptides degraded with carboxypeptidases of different specificities and analyzed by 252-Cf plasma desorption mass spectrometry. Anal Biochem 180: 28–37
Schar M, Bornsen KO, Gassman E (1991) Fast protein sequence determination with matrix-assisted laser desorption and ionization mass spectrometry. Rapid Commun Mass Spectrom 5:319–326
Thiede B, Wittmann-Liebold B, Bienert M, Krause E (1995) MALDI-MS for C-terminal sequence determination of peptides and proteins degraded by carboxypeptidase Y and P. FEBS Lett 357: 65–69
Wang R, Cotter RJ, Meschia JF, Sisodia SS (1992) Determination of the cleavage site of the amyloid precursor protein by plasma desorption mass spectrometry. In: RH Angeletti (ed): Techniques in protein chemistry III. Academic Press, San Diego, 505–513
Wilm M, Mann, M (1996) Analytical properties of the nanoelectrospray ion source. Anal Chem 68: 1–8
Rosnack KJ, Stroh JG (1992) C-terminal sequencing of peptides using electrospray ionization mass spectrometry. Rapid Commun Mass Spectrom 6: 637–640
Smith CE, Duffin KL (1993) Carboxy-terminal protein sequence analysis using carboxypeptidase P and electrospray mass spectrometry. In: RH Angeletti (ed): Techniques in protein chemistry IV. Academic Press, San Diego, 463–470
Patterson DH, Tarr GE, Regnier FE, Martin SA (1995) C-terminal ladder sequencing via matrix-assisted laser desorption mass spectrometry coupled with carboxypeptidase Y time-dependent and concentration-dependent digestions. Anal Chem 67: 3971–3978
Bonetto V, Bergman A-C, Jornvall H, Sillard R (1997) C-terminal sequence analysis of peptides and proteins using carboxypeptidases and mass spectrometry after derivatization of Lys and Cys residues. Anal Chem 69: 1315–1319
Deon C, Prome JC, Prome D, Francina A, Groff P, Kalmes G, Galacteros F, Wajcman H (1997) Combined mass spectrometric methods for the characterization of human hemoglobin variants localized within a T9 peptide: Identification of Hb Villeurbanne a89 (FG1) His →4 Tyr. J Mass Spectrom 32: 880–887
Caprioli RM, Fan T (1986) Peptide sequence analysis using exopeptidases with molecular analysis of the truncated polypeptides by mass spectrometry. Anal Biochem 154: 596–603
Woods AS, Huang AYC, Cotter RJ, Pasternack GR, Pardoll DM, Jaffee EM (1995) Simplified high-sensitivity sequencing of a major histocompatibility complex class I-associated immunoreactive peptide using matrix-assisted laser desorption/ionization mass spectrometry. Anal Biochem 226: 15–25
Thiede B, Salnikow J, Wittmann-Liebold B (1997) C-terminal ladder sequencing by an approach combining chemical degradation with analysis by matrix-assisted-laser-desorption ionization mass spectrometry. Eur J Biochem 244: 750–754
Schlack P, Kumpf W (1926) Ober eine neue Methode zur Ermittelung der Konstitution von Peptiden. Z Physiol Chem 154: 125–170
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2000 Springer Basel AG
About this chapter
Cite this chapter
Bergman, T. (2000). Ladder sequencing. In: Jollès, P., Jörnvall, H. (eds) Proteomics in Functional Genomics. EXS, vol 88. Birkhäuser, Basel. https://doi.org/10.1007/978-3-0348-8458-7_9
Download citation
DOI: https://doi.org/10.1007/978-3-0348-8458-7_9
Publisher Name: Birkhäuser, Basel
Print ISBN: 978-3-0348-9576-7
Online ISBN: 978-3-0348-8458-7
eBook Packages: Springer Book Archive