Analysis of Protein Phosphorylation by a Combination of Elastase Digestion and Neutral Loss Tandem Mass Spectrometry

Schlösser, Andreas; Pipkorn, Ruediger; Bossemeyer, Dirk; Lehmann, Wolf D.

doi:10.1021/ac000826j

Cited by 172 publications

(156 citation statements)

References 30 publications

(35 reference statements)

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“…Schlosser and coworkers have previously shown for phosphorylated peptides that the optimal collision energy is also (linearly) dependent on the size of the peptide [20]. Therefore, only the development of the MNM scan with a collision energy gradient would allow for the collection of mass spectra at each optimal collision energy, producing the most statistically significant autocorrelation interpretation.…”

Section: Methods Developmentmentioning

confidence: 99%

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Multiple neutral loss monitoring (MNM): A multiplexed method for post-translational modification screening

Hoffman

Sniatynski

Rogalski

et al. 2006

J. Am. Soc. Mass Spectrom.

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Post-translational modifications of proteins are involved in determining the activity of proteins and are essential for proper protein function. Current mass spectrometric strategies require one to specify a particular type of modification, in some cases also a particular charge state of a protein or peptide that is to be studied before the actual analysis. Due to these requirements, most of the modifications on proteins are not considered in such an experiment and, thus, a series of similar analyses need to be performed to ensure a more extensive characterization. A novel scan strategy has been developed, multiple neutral loss monitoring (MNM), allowing for the comprehensive screening of post-translational modifications (PTM) on proteins that fragment as neutral losses in a mass spectrometer. MNM method parameters were determined by performing product ion scans on a number of modified peptides over a range of collision energies, providing neutral loss energy profiles and optimal collision energies (OCE) for each modification, supplying valuable information pertaining to the fragmentation of these modifications and the necessary parameters that would be required to obtain the best analysis. As the optimal collision energy was highly dependent on the type of modification and the charge state of the peptide, the MNM scan was operated with a collision energy gradient. Autocorrelation analyses identified the type of modification, and convolution mapping analyses identified the associated peptide. The MNM scan with the new collision energy parameters was successfully applied to a mixture of four modified peptides in a BSA digest. The implementation of this technique will allow for comprehensive screening of all modifications that fragment as neutral losses. (J Am Soc Mass Spectrom 2006, 17, 307-317)

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Section: Methods Developmentmentioning

confidence: 99%

“…This cut off was selected to eliminate the low mass background of the solvent [5,8]. Acceleration voltages of 10,15,20,25, and 30 V were applied from Q0 to Q2 to fragment the transmitted peptides in Q2. The peptides were trapped in Q3, and then sequentially scanned out.…”

Section: Mass Spectrometrymentioning

confidence: 99%

Multiple neutral loss monitoring (MNM): A multiplexed method for post-translational modification screening

Hoffman

Sniatynski

Rogalski

et al. 2006

J. Am. Soc. Mass Spectrom.

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“…Peptides and fragment ions containing phospho-Ser or phospho-Thr can lose phosphoric acid (−98 Da) 8 (Fig. 6).…”

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confidence: 99%

Verification of automated peptide identifications from proteomic tandem mass spectra

2006

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Shotgun proteomics yields tandem mass spectra of peptides that can be identified by database search algorithms. When only a few observed peptides suggest the presence of a protein, establishing the accuracy of the peptide identifications is necessary for accepting or rejecting the protein identification. In this protocol, we describe the properties of peptide identifications that can differentiate legitimately identified peptides from spurious ones. The chemistry of fragmentation, as embodied in the 'mobile proton' and 'pathways in competition' models, informs the process of confirming or rejecting each spectral match. Examples of ion-trap and tandem time-of-flight (TOF/ TOF) mass spectra illustrate these principles of fragmentation.

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“…Precursor ion scanning [14,19], neutral loss scanning [23], and nozzle-skimmer dissociation [9] can determine the presence, absence, and location of peptide phosphorylation based on the unique mass losses associated with phosphorylated peptides. In MALDI-TOF mass spectrometry, postsource decay (PSD) of metastable ions formed in the source region via these characteristic pathways can also be used to determine sites of phosphorylation [17].…”

mentioning

confidence: 99%

Infrared multiphoton dissociation (IRMPD) and collisionally activated dissociationof peptides in a quadrupole ion trapwith selective IRMPD of phosphopeptides

Crowe

Brodbelt

2004

J. Am. Soc. Mass Spectrom.

112

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Dissociation of protonated peptides via infrared multiphoton dissociation (IRMPD) provides more extensive sequence information than is obtained with collisionally activated dissociation (CAD) in a quadrupole ion trap due to the lack of the CAD low m/z cutoff and the ability to form secondary and higher order fragments with the non-resonant photoactivation technique. In addition, IRMPD is shown to be useful for the selective dissociation of phosphopeptides over those which are not phosphorylated because the greater photon absorption efficiency of the phosphorylated peptides leads to their more rapid dissociation. Finally, the selectivity of the IRMPD technique for phosphorylated species in complex mixtures is confirmed with the analysis of a mock peptide mixture and a tryptic digest of ␣-casein. I nvestigations into the protein complement of an organism's genome seek to identify the primary structure of the proteins produced including any modifications that take place. Following transcription, the primary sequence of a protein can undergo modifications such as N-terminal acetylation, formation of disulfide bonds, sulfation, and phosphorylation, all of which affect activity. Arguably, the most important post-translational modification of proteins is the reversible phosphorylation of serine, threonine, and tyrosine residues [1,2]. This covalent modification has regulatory influence over cellular processes such as metabolism, growth, and reproduction [3][4][5]. Because phosphorylation has such an impact on living systems, it is of great interest to be able to determine when, where, and to what extent this type of modification takes place. For this, suitable analytical techniques must be developed and applied. The relative speed and sensitivity of tandem mass spectrometry make it a powerful tool [6 -30] when compared with more conventional methods of phosphoprotein mapping, namely 32 P phosphate labeling of a protein sample followed by purification, enzymatic digestion, chromatographic separation, and Edman sequencing.Collisional activated dissociation (CAD) typically reveals sites of phosphorylation based on a mass loss associated with the cleavage of a phosphate moiety from a peptide ion (Ϫ80 (HPO 3 ) and/or Ϫ98 (H 3 PO 4 ) in positive ion MS/MS) [9,14]. Precursor ion scanning [14,19], neutral loss scanning [23], and nozzle-skimmer dissociation [9] can determine the presence, absence, and location of peptide phosphorylation based on the unique mass losses associated with phosphorylated peptides. In MALDI-TOF mass spectrometry, postsource decay (PSD) of metastable ions formed in the source region via these characteristic pathways can also be used to determine sites of phosphorylation [17]. When a peptide is phosphorylated at a tyrosine residue as opposed to a serine or threonine residue, however, the unique neutral losses are not necessarily observed upon dissociation due to the greater phosphate-tyrosine binding energy and the existence of fewer ␤-elimination pathways than are present in the cases of serine and thr...

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Analysis of Protein Phosphorylation by a Combination of Elastase Digestion and Neutral Loss Tandem Mass Spectrometry

Cited by 172 publications

References 30 publications

Multiple neutral loss monitoring (MNM): A multiplexed method for post-translational modification screening

Multiple neutral loss monitoring (MNM): A multiplexed method for post-translational modification screening

Verification of automated peptide identifications from proteomic tandem mass spectra

Infrared multiphoton dissociation (IRMPD) and collisionally activated dissociationof peptides in a quadrupole ion trapwith selective IRMPD of phosphopeptides

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