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Accurate and Efficient Resolution of Overlapping Isotopic Envelopes in Protein Tandem Mass Spectra.

Xiao K, Yu F, Fang H, Xue B, Liu Y, Tian Z - Sci Rep (2015)

Bottom Line: The relative deviation (RD) of the overall observed experimental abundance of this OIP relative to the summed ideal value is then calculated.Comprehensive data at the protein and proteome levels, high confidence and good reproducibility were achieved.The resolving method reported here can, in principle, be extended to resolve any envelope-type overlapping data for which the corresponding theoretical reference values are available.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Shanghai Key Laboratory of Chemical Assessment and Sustainability, Tongji University, Shanghai 200092, China.

ABSTRACT
It has long been an analytical challenge to accurately and efficiently resolve extremely dense overlapping isotopic envelopes (OIEs) in protein tandem mass spectra to confidently identify proteins. Here, we report a computationally efficient method, called OIE_CARE, to resolve OIEs by calculating the relative deviation between the ideal and observed experimental abundance. In the OIE_CARE method, the ideal experimental abundance of a particular overlapping isotopic peak (OIP) is first calculated for all the OIEs sharing this OIP. The relative deviation (RD) of the overall observed experimental abundance of this OIP relative to the summed ideal value is then calculated. The final individual abundance of the OIP for each OIE is the individual ideal experimental abundance multiplied by 1 + RD. Initial studies were performed using higher-energy collisional dissociation tandem mass spectra on myoglobin (with direct infusion) and the intact E. coli proteome (with liquid chromatographic separation). Comprehensive data at the protein and proteome levels, high confidence and good reproducibility were achieved. The resolving method reported here can, in principle, be extended to resolve any envelope-type overlapping data for which the corresponding theoretical reference values are available.

No MeSH data available.


Related in: MedlinePlus

Interpreted isotopic peaks and abundance of the myoglobin HCD spectra when two incremental combinatorial ions series, b/y and b/y + b/y-NL (including a and a-NL), are searched separately.The error bars arise from three technical replicates. IPs = Isotopic Peaks.
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f5: Interpreted isotopic peaks and abundance of the myoglobin HCD spectra when two incremental combinatorial ions series, b/y and b/y + b/y-NL (including a and a-NL), are searched separately.The error bars arise from three technical replicates. IPs = Isotopic Peaks.

Mentions: Protein-level comprehensiveness, in terms of the percentage of interpreted experimental isotopic peaks and abundance, has been achieved when b, y and their NL ions (including “a” and “a-NL” ions) are included in the database search (Table 1). To evaluate the individual contribution of the various ion series (only b and y ions), the HCD spectra of myoglobin were also independently searched using the same set of tolerance parameters as described above. An increase in the interpreted isotopic peaks and abundance versus these two combinatorial ion series are shown in Fig. 5. The b or y ions are approximately 70% in the number of isotopic peaks and approximately 90% in abundance. This implies that the b or y ions are the most abundant ion series in the HCD spectra of myoglobin. The b or y-NL (including a and a-NL) are approximately 26% in the number of isotopic peaks but approximately 10% in the total abundance. The remaining less than 4% of the isotopic peaks belonged to internal ions or their NL ions. Their total abundance (<1%) is negligible in this case. For comprehensiveness at the proteome level, the identification rate of the E. coli tandem mass spectra from the three technical replicate RPLC-MSMS runs is 73.3 ± 3.4%. The identification rate is defined as the total number of PrSMs from the dataset divided by the total number of MS/MS spectra between the first and last PrSMs. Here the MS/MS spectra were acquired only for precursors with ≥5 or with unassigned charge states. This rate could be further improved by additional search of the proteolytic peptidome, as well as by more comprehensive annotation of PTMs. The current protein-annotation rate in terms of ‘MOD_RES’ in the flat text file was only 5.2%. These extra utilities for ProteinGoggle are under development.


Accurate and Efficient Resolution of Overlapping Isotopic Envelopes in Protein Tandem Mass Spectra.

Xiao K, Yu F, Fang H, Xue B, Liu Y, Tian Z - Sci Rep (2015)

Interpreted isotopic peaks and abundance of the myoglobin HCD spectra when two incremental combinatorial ions series, b/y and b/y + b/y-NL (including a and a-NL), are searched separately.The error bars arise from three technical replicates. IPs = Isotopic Peaks.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4593959&req=5

f5: Interpreted isotopic peaks and abundance of the myoglobin HCD spectra when two incremental combinatorial ions series, b/y and b/y + b/y-NL (including a and a-NL), are searched separately.The error bars arise from three technical replicates. IPs = Isotopic Peaks.
Mentions: Protein-level comprehensiveness, in terms of the percentage of interpreted experimental isotopic peaks and abundance, has been achieved when b, y and their NL ions (including “a” and “a-NL” ions) are included in the database search (Table 1). To evaluate the individual contribution of the various ion series (only b and y ions), the HCD spectra of myoglobin were also independently searched using the same set of tolerance parameters as described above. An increase in the interpreted isotopic peaks and abundance versus these two combinatorial ion series are shown in Fig. 5. The b or y ions are approximately 70% in the number of isotopic peaks and approximately 90% in abundance. This implies that the b or y ions are the most abundant ion series in the HCD spectra of myoglobin. The b or y-NL (including a and a-NL) are approximately 26% in the number of isotopic peaks but approximately 10% in the total abundance. The remaining less than 4% of the isotopic peaks belonged to internal ions or their NL ions. Their total abundance (<1%) is negligible in this case. For comprehensiveness at the proteome level, the identification rate of the E. coli tandem mass spectra from the three technical replicate RPLC-MSMS runs is 73.3 ± 3.4%. The identification rate is defined as the total number of PrSMs from the dataset divided by the total number of MS/MS spectra between the first and last PrSMs. Here the MS/MS spectra were acquired only for precursors with ≥5 or with unassigned charge states. This rate could be further improved by additional search of the proteolytic peptidome, as well as by more comprehensive annotation of PTMs. The current protein-annotation rate in terms of ‘MOD_RES’ in the flat text file was only 5.2%. These extra utilities for ProteinGoggle are under development.

Bottom Line: The relative deviation (RD) of the overall observed experimental abundance of this OIP relative to the summed ideal value is then calculated.Comprehensive data at the protein and proteome levels, high confidence and good reproducibility were achieved.The resolving method reported here can, in principle, be extended to resolve any envelope-type overlapping data for which the corresponding theoretical reference values are available.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Shanghai Key Laboratory of Chemical Assessment and Sustainability, Tongji University, Shanghai 200092, China.

ABSTRACT
It has long been an analytical challenge to accurately and efficiently resolve extremely dense overlapping isotopic envelopes (OIEs) in protein tandem mass spectra to confidently identify proteins. Here, we report a computationally efficient method, called OIE_CARE, to resolve OIEs by calculating the relative deviation between the ideal and observed experimental abundance. In the OIE_CARE method, the ideal experimental abundance of a particular overlapping isotopic peak (OIP) is first calculated for all the OIEs sharing this OIP. The relative deviation (RD) of the overall observed experimental abundance of this OIP relative to the summed ideal value is then calculated. The final individual abundance of the OIP for each OIE is the individual ideal experimental abundance multiplied by 1 + RD. Initial studies were performed using higher-energy collisional dissociation tandem mass spectra on myoglobin (with direct infusion) and the intact E. coli proteome (with liquid chromatographic separation). Comprehensive data at the protein and proteome levels, high confidence and good reproducibility were achieved. The resolving method reported here can, in principle, be extended to resolve any envelope-type overlapping data for which the corresponding theoretical reference values are available.

No MeSH data available.


Related in: MedlinePlus