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Analytical utility of mass spectral binning in proteomic experiments by SPectral Immonium Ion Detection (SPIID).

Kelstrup CD, Frese C, Heck AJ, Olsen JV, Nielsen ML - Mol. Cell Proteomics (2014)

Bottom Line: Although such ions offer tremendous analytical advantages, algorithms to decipher MS/MS spectra for the presence of diagnostic ions in an unbiased manner are currently lacking.To benchmark the software tool, we analyzed large higher-energy collisional activation dissociation datasets of samples containing phosphorylation, ubiquitylation, SUMOylation, formylation, and lysine acetylation.Using the developed software tool, we were able to identify known diagnostic ions by comparing histograms of modified and unmodified peptide spectra.

View Article: PubMed Central - PubMed

Affiliation: From the ‡Department of Proteomics, The Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, Faculty of Health Sciences, DK-2200 Copenhagen, Denmark;

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Related in: MedlinePlus

A, experimental description of spectral binning by SPIID. All acquired MS/MS spectra are binned into mass bins, yielding a histogram in which analogous fragment ions such as immonium ions are located in same mass bins. B, histogram representing frequently occurring ions from a tryptic digest, primarily represented by amino-specific immonium ions and tryptic y1 ions. C, very low bin sizes can be used for HCD-generated MS/MS spectra, as shown for the mass accuracy of the lysine y1 ion by spectral binning. To demonstrate this, we specifically for this analysis binned with a mass tolerance of ±0.0005 Da. D, number of theoretical chemical isomers at various mass accuracies. E, list of the most commonly observed tryptic fragment ions measured with high resolution after HCD fragmentation.
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Figure 1: A, experimental description of spectral binning by SPIID. All acquired MS/MS spectra are binned into mass bins, yielding a histogram in which analogous fragment ions such as immonium ions are located in same mass bins. B, histogram representing frequently occurring ions from a tryptic digest, primarily represented by amino-specific immonium ions and tryptic y1 ions. C, very low bin sizes can be used for HCD-generated MS/MS spectra, as shown for the mass accuracy of the lysine y1 ion by spectral binning. To demonstrate this, we specifically for this analysis binned with a mass tolerance of ±0.0005 Da. D, number of theoretical chemical isomers at various mass accuracies. E, list of the most commonly observed tryptic fragment ions measured with high resolution after HCD fragmentation.

Mentions: The basic principle of the approach relies on the knowledge that immonium ions and PTM-specific diagnostic ions will appear at the same m/z in MS/MS spectra, whereas sequence-specific fragment ions appear at discrete m/z values in the investigated mass range. As a result, creating a histogram of all observed fragmentation ions across a number of MS/MS spectra will result in common ions (immonium and diagnostic ions) clustering into unique mass bins, whereas backbone fragment ions (b- and y-ions) will scatter across the entire bin range (Fig. 1A). To demonstrate the spectral mass binning approach, we binned MS/MS spectra derived from a large-scale analysis of standard HeLa lysates (23). The generated histogram depicts all commonly occurring fragment ions throughout the binned MS/MS range (Fig. 1B). The strongest peaks present in the histogram correspond to known immonium ions for amino acids such as lysine/glutamine (m/z 101.0709), histidine (m/z 110.0713), phenylalanine (m/z 120.0808), tyrosine (m/z 136.0757), and tryptophan (m/z 159.0917). Because the dataset is derived from a HeLa lysate digested with trypsin, the majority of peptide sequences contain either a lysine or an arginine at the C-terminal position (36). Following this, the spectral binning approach identified the trypsin-specific y1 ions for lysine (m/z 147.1128) and arginine (m/z 175.1190). Notably, the abundance of these two mass bins was very similar, confirming that an equal share of the investigated MS/MS spectra harbored either a lysine or an arginine y1 ion.


Analytical utility of mass spectral binning in proteomic experiments by SPectral Immonium Ion Detection (SPIID).

Kelstrup CD, Frese C, Heck AJ, Olsen JV, Nielsen ML - Mol. Cell Proteomics (2014)

A, experimental description of spectral binning by SPIID. All acquired MS/MS spectra are binned into mass bins, yielding a histogram in which analogous fragment ions such as immonium ions are located in same mass bins. B, histogram representing frequently occurring ions from a tryptic digest, primarily represented by amino-specific immonium ions and tryptic y1 ions. C, very low bin sizes can be used for HCD-generated MS/MS spectra, as shown for the mass accuracy of the lysine y1 ion by spectral binning. To demonstrate this, we specifically for this analysis binned with a mass tolerance of ±0.0005 Da. D, number of theoretical chemical isomers at various mass accuracies. E, list of the most commonly observed tryptic fragment ions measured with high resolution after HCD fragmentation.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4125726&req=5

Figure 1: A, experimental description of spectral binning by SPIID. All acquired MS/MS spectra are binned into mass bins, yielding a histogram in which analogous fragment ions such as immonium ions are located in same mass bins. B, histogram representing frequently occurring ions from a tryptic digest, primarily represented by amino-specific immonium ions and tryptic y1 ions. C, very low bin sizes can be used for HCD-generated MS/MS spectra, as shown for the mass accuracy of the lysine y1 ion by spectral binning. To demonstrate this, we specifically for this analysis binned with a mass tolerance of ±0.0005 Da. D, number of theoretical chemical isomers at various mass accuracies. E, list of the most commonly observed tryptic fragment ions measured with high resolution after HCD fragmentation.
Mentions: The basic principle of the approach relies on the knowledge that immonium ions and PTM-specific diagnostic ions will appear at the same m/z in MS/MS spectra, whereas sequence-specific fragment ions appear at discrete m/z values in the investigated mass range. As a result, creating a histogram of all observed fragmentation ions across a number of MS/MS spectra will result in common ions (immonium and diagnostic ions) clustering into unique mass bins, whereas backbone fragment ions (b- and y-ions) will scatter across the entire bin range (Fig. 1A). To demonstrate the spectral mass binning approach, we binned MS/MS spectra derived from a large-scale analysis of standard HeLa lysates (23). The generated histogram depicts all commonly occurring fragment ions throughout the binned MS/MS range (Fig. 1B). The strongest peaks present in the histogram correspond to known immonium ions for amino acids such as lysine/glutamine (m/z 101.0709), histidine (m/z 110.0713), phenylalanine (m/z 120.0808), tyrosine (m/z 136.0757), and tryptophan (m/z 159.0917). Because the dataset is derived from a HeLa lysate digested with trypsin, the majority of peptide sequences contain either a lysine or an arginine at the C-terminal position (36). Following this, the spectral binning approach identified the trypsin-specific y1 ions for lysine (m/z 147.1128) and arginine (m/z 175.1190). Notably, the abundance of these two mass bins was very similar, confirming that an equal share of the investigated MS/MS spectra harbored either a lysine or an arginine y1 ion.

Bottom Line: Although such ions offer tremendous analytical advantages, algorithms to decipher MS/MS spectra for the presence of diagnostic ions in an unbiased manner are currently lacking.To benchmark the software tool, we analyzed large higher-energy collisional activation dissociation datasets of samples containing phosphorylation, ubiquitylation, SUMOylation, formylation, and lysine acetylation.Using the developed software tool, we were able to identify known diagnostic ions by comparing histograms of modified and unmodified peptide spectra.

View Article: PubMed Central - PubMed

Affiliation: From the ‡Department of Proteomics, The Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, Faculty of Health Sciences, DK-2200 Copenhagen, Denmark;

Show MeSH
Related in: MedlinePlus