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Illuminating the dark matter of shotgun proteomics Owen S Skinner & Neil L Kelleher
npg
© 2015 Nature America, Inc. All rights reserved.
Many of the unassignable spectra in proteomics data represent peptides with post-translational modifications. In shotgun proteomics, proteins are enzymat ically digested into peptides that are fed into a highly automated pipeline for chromatography and mass spectrometry (MS) analysis1. When applied to complex mixtures of proteins from cells or tissues, the technique yields tens or hundreds of thousands of peptide sequences, only a minority of which can be mapped to their proteins of origin. Despite the efforts of many researchers, the identity and origin of the unassigned peptides have remained a mystery. In this issue, Chick et al.2 show that many peptides escape identification because they have unexpected post-translational modifications (Fig. 1). Covalent modification of a protein’s amino acid residues often changes its mass. Because shotgun proteomics relies on massbased search techniques, peptides that have unexpected mass differences often remain unidentified (Fig. 1b, traditional search). Modifications can occur during biological processes or during sample preparation, especially during enzymatic digestion. One analysis has suggested that every residue of every peptide is modified at least 1% of the time3. Given the large numbers of peptides observed in data sets, manual validation of each unidentified spectrum to characterize potential modifications is not feasible, creating a need for automated peptide identification through database searching. The search method developed by Chick et al.2 is simple but paradoxical. Although the peptide mass data that they collected are very accurate, they used a wide mass tolerance when matching their spectra to Owen S. Skinner and Neil L. Kelleher are at the Department of Chemistry, Northwestern University, Evanston, Illinois, USA. Neil L. Kelleher is also at the Department of Molecular Biosciences, Northwestern University, Evanston, Illinois, USA. e-mail: [email protected]
peptide databases. They calculated that 93% of known protein modifications result in mass shifts of ±500 Da or less. Therefore, allowing for these shifts greatly improves the ability to identify peptides (Fig. 1). The idea of increasing mass tolerances in proteomics searches is not new. ‘Top-down’ proteomics, which interrogates intact proteins directly without proteolytic degradation, has used large search windows to good effect for some time4. The method has also
a
been implemented in shotgun proteomics and was pilot tested on ~10,000 peptides3. However, no previous study has achieved the scale and vision of the present work. Starting with 396,736 peptides that they identified in proteins from human HEK 293 cells by a traditional search method, Chick et al.2 were able to find an additional ~184,000 peptides by using their ‘open’ search strategy. The spectacular 46% increase in identifications required no additional data 1,000 Da 10 p.p.m.
125.900 Da
Mass
b
125.900 Da
Traditional search
Open search
125.900 Da > 10 p.p.m.
125.900 Da < 500 Da
+ Modification Figure 1 Illustration of the benefits of an open search strategy, as used to identify 184,000 modified peptides. (a) The intact mass spectrum of a peptide is measured at a mass accuracy of
Illuminating the dark matter of shotgun proteomics Owen S Skinner & Neil L Kelleher
npg
© 2015 Nature America, Inc. All rights reserved.
Many of the unassignable spectra in proteomics data represent peptides with post-translational modifications. In shotgun proteomics, proteins are enzymat ically digested into peptides that are fed into a highly automated pipeline for chromatography and mass spectrometry (MS) analysis1. When applied to complex mixtures of proteins from cells or tissues, the technique yields tens or hundreds of thousands of peptide sequences, only a minority of which can be mapped to their proteins of origin. Despite the efforts of many researchers, the identity and origin of the unassigned peptides have remained a mystery. In this issue, Chick et al.2 show that many peptides escape identification because they have unexpected post-translational modifications (Fig. 1). Covalent modification of a protein’s amino acid residues often changes its mass. Because shotgun proteomics relies on massbased search techniques, peptides that have unexpected mass differences often remain unidentified (Fig. 1b, traditional search). Modifications can occur during biological processes or during sample preparation, especially during enzymatic digestion. One analysis has suggested that every residue of every peptide is modified at least 1% of the time3. Given the large numbers of peptides observed in data sets, manual validation of each unidentified spectrum to characterize potential modifications is not feasible, creating a need for automated peptide identification through database searching. The search method developed by Chick et al.2 is simple but paradoxical. Although the peptide mass data that they collected are very accurate, they used a wide mass tolerance when matching their spectra to Owen S. Skinner and Neil L. Kelleher are at the Department of Chemistry, Northwestern University, Evanston, Illinois, USA. Neil L. Kelleher is also at the Department of Molecular Biosciences, Northwestern University, Evanston, Illinois, USA. e-mail: [email protected]
peptide databases. They calculated that 93% of known protein modifications result in mass shifts of ±500 Da or less. Therefore, allowing for these shifts greatly improves the ability to identify peptides (Fig. 1). The idea of increasing mass tolerances in proteomics searches is not new. ‘Top-down’ proteomics, which interrogates intact proteins directly without proteolytic degradation, has used large search windows to good effect for some time4. The method has also
a
been implemented in shotgun proteomics and was pilot tested on ~10,000 peptides3. However, no previous study has achieved the scale and vision of the present work. Starting with 396,736 peptides that they identified in proteins from human HEK 293 cells by a traditional search method, Chick et al.2 were able to find an additional ~184,000 peptides by using their ‘open’ search strategy. The spectacular 46% increase in identifications required no additional data 1,000 Da 10 p.p.m.
125.900 Da
Mass
b
125.900 Da
Traditional search
Open search
125.900 Da > 10 p.p.m.
125.900 Da < 500 Da
+ Modification Figure 1 Illustration of the benefits of an open search strategy, as used to identify 184,000 modified peptides. (a) The intact mass spectrum of a peptide is measured at a mass accuracy of
