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Protein sequencing by mass analysis of polypeptide ladders after controlled protein hydrolysis

Nature Biotechnology volume 22, pages 1291–1296 (2004)Cite this article

Abstract

The characterization of protein modifications is essential for the study of protein function using functional genomic and proteomic approaches. However, current techniques are not efficient in determining protein modifications. We report an approach for sequencing proteins and determining modifications with high speed, sensitivity and specificity. We discovered that a protein could be readily acid-hydrolyzed within 1 min by exposure to microwave irradiation to form, predominantly, two series of polypeptide ladders containing either the N- or C-terminal amino acid of the protein, respectively. Mass spectrometric analysis of the hydrolysate produced a simple mass spectrum consisting of peaks exclusively from these polypeptide ladders, allowing direct reading of amino acid sequence and modifications of the protein. As examples, we applied this technique to determine protein phosphorylation sites as well as the sequences and several previously unknown modifications of 28 small proteins isolated from Escherichia coli K12 cells. This technique can potentially be automated for large-scale protein annotation.

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Figure 1: Schematic of the mass analysis of polypeptide-ladders (MAP) sequencing technique.
Figure 2: MALDI spectra of 1 pmol of cytochrome c.
Figure 3: MALDI spectra of an HPLC-fractionated sample from an E. coli K12 extract.
Figure 4: MALDI spectrum (in red) of a sample containing 85% α-S1-casein and MALDI spectrum (in black) of a sample containing 85% dephosphorylated α-S1-casein.

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Acknowledgements

This work was supported by the Natural Sciences and Engineering Research Council of Canada, Genome Canada through Genome Prairie's Enabling Technologies Project, Canada Foundation for Innovation, The Protein Engineering Network of Centres of Excellence, and Alberta Cancer Board. We thank Andrew Shaw (Cross Cancer Institute, Alberta) for helpful comments on the manuscript.

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Authors and Affiliations

  1. Department of Chemistry, University of Alberta, Chemistry Center W3-39, Edmonton, T6G 2G2, Alberta, Canada

    Hongying Zhong, Ying Zhang, Zhihui Wen & Liang Li

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  1. Hongying Zhong
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Correspondence to Liang Li.

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Supplementary information

Supplementary Fig. 1

Expanded MALDI spectra of Figure 2 in the m/z range from 2,500 to 6,000. (PDF 121 kb)

Supplementary Fig. 2

MALDI spectra of 1 pmol of lysozyme. (PDF 166 kb)

Supplementary Fig. 3

MALDI spectra of an HPLC-fractionated sample from an E. coli K12 extrac (PDF 44 kb)

Supplementary Fig. 4

Expanded MALDI spectra of an HPLC-fractionated sample from an E. coli K12 extract after mixing with 6M HCl and applying microwave irradiation for 1 min. (PDF 97 kb)

Supplementary Fig. 5

MAP sequencing of human ubiquitin using different amounts of starting materials ranging from 7 fmol to 7 pmol. (PDF 67 kb)

Supplementary Fig. 6

Effect of SDS on acid hydrolysis with MI. (PDF 181 kb)

Supplementary Table 1

List of proteins characterized by the MAP sequencing technique. (PDF 17 kb)

Supplementary Note (PDF 91 kb)

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Zhong, H., Zhang, Y., Wen, Z. et al. Protein sequencing by mass analysis of polypeptide ladders after controlled protein hydrolysis. Nat Biotechnol 22, 1291–1296 (2004). https://doi.org/10.1038/nbt1011

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