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A high confidence, manually validated human blood plasma protein reference set.

Schenk S, Schoenhals GJ, de Souza G, Mann M - BMC Med Genomics (2008)

Bottom Line: Both instruments allow the measurement of peptide masses in the low ppm range.Furthermore, we employed a statistical score that allows database peptide identification searching using the products of two consecutive stages of tandem mass spectrometry (MS3).The combination of MS3 with very high mass accuracy in the parent peptide allows peptide identification with orders of magnitude more confidence than that typically achieved.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Bioinformatics, University of Southern Denmark, Campusvej 55, 5230 Odense M,Denmark. sschenk@bmb.sdu.dk

ABSTRACT

Background: The immense diagnostic potential of human plasma has prompted great interest and effort in cataloging its contents, exemplified by the Human Proteome Organization (HUPO) Plasma Proteome Project (PPP) pilot project. Due to challenges in obtaining a reliable blood plasma protein list, HUPO later re-analysed their own original dataset with a more stringent statistical treatment that resulted in a much reduced list of high confidence (at least 95%) proteins compared with their original findings. In order to facilitate the discovery of novel biomarkers in the future and to realize the full diagnostic potential of blood plasma, we feel that there is still a need for an ultra-high confidence reference list (at least 99% confidence) of blood plasma proteins.

Methods: To address the complexity and dynamic protein concentration range of the plasma proteome, we employed a linear ion-trap-Fourier transform (LTQ-FT) and a linear ion trap-Orbitrap (LTQ-Orbitrap) for mass spectrometry (MS) analysis. Both instruments allow the measurement of peptide masses in the low ppm range. Furthermore, we employed a statistical score that allows database peptide identification searching using the products of two consecutive stages of tandem mass spectrometry (MS3). The combination of MS3 with very high mass accuracy in the parent peptide allows peptide identification with orders of magnitude more confidence than that typically achieved.

Results: Herein we established a high confidence set of 697 blood plasma proteins and achieved a high 'average sequence coverage' of more than 14 peptides per protein and a median of 6 peptides per protein. All proteins annotated as belonging to the immunoglobulin family as well as all hypothetical proteins whose peptides completely matched immunoglobulin sequences were excluded from this protein list. We also compared the results of using two high-end MS instruments as well as the use of various peptide and protein separation approaches. Furthermore, we characterized the plasma proteins using cellular localization information, as well as comparing our list of proteins to data from other sources, including the HUPO PPP dataset.

Conclusion: Superior instrumentation combined with rigorous validation criteria gave rise to a set of 697 plasma proteins in which we have very high confidence, demonstrated by an exceptionally low false peptide identification rate of 0.29%.

No MeSH data available.


Related in: MedlinePlus

Venn diagram representations of comparisons between pairs of individual plasma experiments. Individual experiments are represented by circles, with compared experiment pairs being depicted by overlapping sets of two circles. The number of proteins identified in both members of a compared set of experiments is given in the intersection region of the circles. The number of proteins that are unique to an individual experiment is shown outside of the intersection region, along with the total number of proteins identified for that individual experiment, shown in parentheses. The plasma experiment represented by each circle is represented at the top of the respective circle, outside of the intersection region (panel A, for example, shows a comparison of experiments 01_MS2, designated "01'', and 04_MS2_prec, designated "04''). The experimental conditions used in each experiment can be found in Table 1. Panel F shows a comparison between all 7 experiments performed on the FT and the last experiment which was performed on the Orbitrap.
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Figure 6: Venn diagram representations of comparisons between pairs of individual plasma experiments. Individual experiments are represented by circles, with compared experiment pairs being depicted by overlapping sets of two circles. The number of proteins identified in both members of a compared set of experiments is given in the intersection region of the circles. The number of proteins that are unique to an individual experiment is shown outside of the intersection region, along with the total number of proteins identified for that individual experiment, shown in parentheses. The plasma experiment represented by each circle is represented at the top of the respective circle, outside of the intersection region (panel A, for example, shows a comparison of experiments 01_MS2, designated "01'', and 04_MS2_prec, designated "04''). The experimental conditions used in each experiment can be found in Table 1. Panel F shows a comparison between all 7 experiments performed on the FT and the last experiment which was performed on the Orbitrap.

Mentions: All of the protein validation rules that we applied to the combined set of 697 proteins were also applied to each individual experimental dataset before comparison. In the course of analysing the data from the different experimental treatments listed in Table 1, we noted some interesting observations. In performing the comparisons, we looked at the total number of proteins identified and those found to be in common between experiment pairs (Figure 6). Furthermore, the valid, non-redundant peptide distribution vs. proteins identified (Figure 7), as well as the MW distributions (Figure 8) for the different experiments were investigated.


A high confidence, manually validated human blood plasma protein reference set.

Schenk S, Schoenhals GJ, de Souza G, Mann M - BMC Med Genomics (2008)

Venn diagram representations of comparisons between pairs of individual plasma experiments. Individual experiments are represented by circles, with compared experiment pairs being depicted by overlapping sets of two circles. The number of proteins identified in both members of a compared set of experiments is given in the intersection region of the circles. The number of proteins that are unique to an individual experiment is shown outside of the intersection region, along with the total number of proteins identified for that individual experiment, shown in parentheses. The plasma experiment represented by each circle is represented at the top of the respective circle, outside of the intersection region (panel A, for example, shows a comparison of experiments 01_MS2, designated "01'', and 04_MS2_prec, designated "04''). The experimental conditions used in each experiment can be found in Table 1. Panel F shows a comparison between all 7 experiments performed on the FT and the last experiment which was performed on the Orbitrap.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2563020&req=5

Figure 6: Venn diagram representations of comparisons between pairs of individual plasma experiments. Individual experiments are represented by circles, with compared experiment pairs being depicted by overlapping sets of two circles. The number of proteins identified in both members of a compared set of experiments is given in the intersection region of the circles. The number of proteins that are unique to an individual experiment is shown outside of the intersection region, along with the total number of proteins identified for that individual experiment, shown in parentheses. The plasma experiment represented by each circle is represented at the top of the respective circle, outside of the intersection region (panel A, for example, shows a comparison of experiments 01_MS2, designated "01'', and 04_MS2_prec, designated "04''). The experimental conditions used in each experiment can be found in Table 1. Panel F shows a comparison between all 7 experiments performed on the FT and the last experiment which was performed on the Orbitrap.
Mentions: All of the protein validation rules that we applied to the combined set of 697 proteins were also applied to each individual experimental dataset before comparison. In the course of analysing the data from the different experimental treatments listed in Table 1, we noted some interesting observations. In performing the comparisons, we looked at the total number of proteins identified and those found to be in common between experiment pairs (Figure 6). Furthermore, the valid, non-redundant peptide distribution vs. proteins identified (Figure 7), as well as the MW distributions (Figure 8) for the different experiments were investigated.

Bottom Line: Both instruments allow the measurement of peptide masses in the low ppm range.Furthermore, we employed a statistical score that allows database peptide identification searching using the products of two consecutive stages of tandem mass spectrometry (MS3).The combination of MS3 with very high mass accuracy in the parent peptide allows peptide identification with orders of magnitude more confidence than that typically achieved.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Bioinformatics, University of Southern Denmark, Campusvej 55, 5230 Odense M,Denmark. sschenk@bmb.sdu.dk

ABSTRACT

Background: The immense diagnostic potential of human plasma has prompted great interest and effort in cataloging its contents, exemplified by the Human Proteome Organization (HUPO) Plasma Proteome Project (PPP) pilot project. Due to challenges in obtaining a reliable blood plasma protein list, HUPO later re-analysed their own original dataset with a more stringent statistical treatment that resulted in a much reduced list of high confidence (at least 95%) proteins compared with their original findings. In order to facilitate the discovery of novel biomarkers in the future and to realize the full diagnostic potential of blood plasma, we feel that there is still a need for an ultra-high confidence reference list (at least 99% confidence) of blood plasma proteins.

Methods: To address the complexity and dynamic protein concentration range of the plasma proteome, we employed a linear ion-trap-Fourier transform (LTQ-FT) and a linear ion trap-Orbitrap (LTQ-Orbitrap) for mass spectrometry (MS) analysis. Both instruments allow the measurement of peptide masses in the low ppm range. Furthermore, we employed a statistical score that allows database peptide identification searching using the products of two consecutive stages of tandem mass spectrometry (MS3). The combination of MS3 with very high mass accuracy in the parent peptide allows peptide identification with orders of magnitude more confidence than that typically achieved.

Results: Herein we established a high confidence set of 697 blood plasma proteins and achieved a high 'average sequence coverage' of more than 14 peptides per protein and a median of 6 peptides per protein. All proteins annotated as belonging to the immunoglobulin family as well as all hypothetical proteins whose peptides completely matched immunoglobulin sequences were excluded from this protein list. We also compared the results of using two high-end MS instruments as well as the use of various peptide and protein separation approaches. Furthermore, we characterized the plasma proteins using cellular localization information, as well as comparing our list of proteins to data from other sources, including the HUPO PPP dataset.

Conclusion: Superior instrumentation combined with rigorous validation criteria gave rise to a set of 697 plasma proteins in which we have very high confidence, demonstrated by an exceptionally low false peptide identification rate of 0.29%.

No MeSH data available.


Related in: MedlinePlus