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The abc's (and xyz's) of peptide sequencing

Nature Reviews Molecular Cell Biology volume 5, pages 699–711 (2004)Cite this article

Key Points

  • For mass spectrometry (MS) analysis, the proteins of interest are proteolytically digested — the resulting peptides are easier to handle, easier to sequence and have better detection efficiencies than intact proteins.

  • Thousands of peptides can be introduced to the mass spectrometer through 'on-line' capillary chromatography. Using MS, their masses can be measured and they can be fragmented to yield partial amino-acid-sequence information (tandem MS).

  • Powerful algorithms can match the data from tandem MS against possible peptide sequences in amino-acid databases. The resulting protein probability scores need to be studied carefully to avoid over-interpreting the identification results, and unbiased statistical techniques are now helping to address such problems.

  • Protein modifications are amenable to MS analysis, as these modifications normally induce mass shifts. However, due to the substoichiometric amounts of protein modifications, selective enrichment and detection methods are usually necessary and there is no guarantee that the complete primary structure of the protein will be covered.

  • Proteins can be quantified by MS using stable-isotope labels. If the relative abundance of a protein in two samples is to be compared, labelling with stable isotopes is the method of choice. The use of isotopically labelled internal standards is recommended for absolute quantification. However, peak intensities and the number of peptides that are observed during a liquid-chromatography–MS experiment (versus the number of theoretically observable peptides that can be derived from the protein of interest) can also be used to estimate protein abundance.

  • There has been great progress in the proteomic analysis of multiprotein complexes and subcellular organelles. However, routine, in-depth proteome analyses of whole-cell lysates, tissue samples and plasma still elude the dynamic-range capabilities and sensitivity of the instruments that are available at present.

Abstract

Proteomics is an increasingly powerful and indispensable technology in molecular cell biology. It can be used to identify the components of small protein complexes and large organelles, to determine post-translational modifications and in sophisticated functional screens. The key — but little understood — technology in mass-spectrometry-based proteomics is peptide sequencing, which we describe and review here in an easily accessible format.

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Figure 1: The mass-spectrometry/proteomic experiment.
Figure 2: The liquid-chromatography–tandem-mass-spectrometry experiment.
Figure 3: Mass-spectrometry traces.
Figure 4: Techniques for the relative quantification of protein populations.

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Acknowledgements

We thank our colleagues at the Center for Experimental BioInformatics (CEBI) and Harvard Medical School for fruitful discussions and for critically reading the manuscript. Work at the CEBI is supported by generous grants from the Danish National Research Foundation (Grundforskningsfond) and the European Union sixth framework programme.

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Authors and Affiliations

  1. Department of Systems Biology, Harvard Medical School, 240 Longwood Avenue, Boston, 02115, Massachusetts, USA

    Hanno Steen

  2. Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, Odense M, DK-5230, Denmark

    Matthias Mann

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Correspondence to Matthias Mann.

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FURTHER INFORMATION

American Society for Mass Spectrometry (ASMS)

Center for Experimental BioInformatics (CEBI)

Human Proteome Organisation (HUPO)

Institute for Systems Biology

SpectroscopyNOW.com, Proteomics

Supplementary material on peptide validation

The Nobel Prize in Chemistry 2002 (for mass spectrometry)

Glossary

MICROSCALE CAPILLARY HPLC COLUMN

High-performance liquid chromatography (HPLC) columns have inner diameters of 50–150 μm and a reversed-phase stationary phase. Reversed phase means that the surface is made using long hydrophobic alkyl chains, so they retain hydrophobic compounds better than hydrophilic ones.

m/z RATIO

(mass-to-charge ratio). Mass spectrometry does not measure the mass of molecules, but instead measures their m/z value. Electrospray ionization, in particular, generates ions with multiple charges, such that the observed m/z value has to be multiplied by z and corrected for the number of attached protons (which equals z) to calculate the molecular weight of a particular peptide.

QUADRUPOLE MASS SPECTROMETER

A mass-selective 'quadrupole section' only allows the passage of ions that have a specific mass to charge (m/z) value by applying a particular sinusoidal potential. Stepping through the m/z range by applying different potentials and detecting the ions that pass through at each m/z value generates the mass spectrum.

TIME OF FLIGHT (TOF) MASS SPECTROMETER

This mass analyser is based on the time it takes ions to travel through an electric-field-free flight tube. In the ion source, all the ions are accelerated to the same kinetic energy. As kinetic energy is a function of mass, the lighter ions fly faster than the heavier ones and therefore reach the detector sooner.

QUADRUPOLE 'ION TRAPS'

In ion traps, the ions are first caught (trapped) in a dynamic electric field and are then sequentially — according to their mass to charge (m/z) value — ejected onto the detector with the help of another electric field. Trapped ions can also be isolated and fragmented within the trap.

DALTON

(Da). The unit of the mass scale, which is defined as one twelfth of the mass of the mono-isotopic form of carbon, 12C (1 Da = 1.6605 × 10−27 kg). Other commonly, but not necessarily correctly, used units of relevance to mass spectrometry are the amu (an atomic mass unit that is based on 16O), the Thomson (the proposed unit for the mass to charge (m/z) scale) and the u ('unit', which is the same as Da).

DE NOVO SEQUENCING

Deriving the amino-acid sequence (primary structure) of a peptide solely from the mass-spectrometry, peptide-fragmentation data (that is, without using databases).

TOTAL ION CURRENT

The sum of all the ion signals in a mass spectrum as a function of elution time.

EXTRACTED ION CURRENT

The sum of the ion signal for a particular mass to charge (m/z) value — that is, for a particular peptide-ion species.

IONIZATION EFFICIENCY

The fraction of peptides in solution that is converted to peptide ions in the gas phase.

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Steen, H., Mann, M. The abc's (and xyz's) of peptide sequencing. Nat Rev Mol Cell Biol 5, 699–711 (2004). https://doi.org/10.1038/nrm1468

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