Fragmentation of phosphopeptides in an ion trap mass spectrometer

Recently, we reported a fast on-line alkaline micro-liquid chromatography/electrosprayatmospheric pressure ionization/collision-induced dissociation/mass spectrometric approach for sensitive phosphopeptide screening of a tryptic digested protein and subsequent characterization of the identified phosphopeptide. Based on this study, we now applied an improved method for the identification of phosphorylation sites in insulin receptor substrate 1, an important mediator in insulin signal transduction which was phosphorylated in vitro by protein kinase C-. The approach consists of an on-line alkaline negative-ion micro-liquid chromatography/electrospray-atmospheric pressure ionization/collision-induced dissociation/mass spectrometric hybrid scan experiment using a triple-quadrupole mass spectrometer with fractionation and subsequent off-line nanoES-MS (ion trap) analysis of the phosphopeptide-containing fractions. During the liquid chromatography (LC)/ES-MS experiment, the phosphopeptides of the enzymatic digest mixture of the studied insulin receptor substrate 1 fragment were detected under high skimmer potential (API-CID) using phosphorylationspecific m/z 79 marker ions as well as the intact m/z-values of the peptides which were recorded under low skimmer potential. Subsequently, the targeted fractions were analyzed by off-line nanoES-MS/MS and MS 3 . Using this approach, serine 318 was clearly identified as a major in vitro protein kinase C-phosphorylation site in the insulin receptor substrate Ϫ1 fragment. Together, our results indicate that the applied strategy is useful for unequivocal and fast analysis of phosphorylation sites in low abundant signaling proteins. (J Am Soc Mass Spectrom 2003, 14, 401-405)

Section: Resultsmentioning

confidence: 99%

Identification of an in vitro insulin receptor substrate-1 phosphorylation site by negative-ion μLC/ES-API-CID-MS hybrid scan technique

Beck

Moeschel

Deeg

et al. 2003

“…Previous work by others also noted a 98 Da loss from phosphotyrosine peptides [11,12,16], however, the reasons for this 98 Da loss is unclear. In order to explore fragmentation of the phosphate group from tyrosine, we employed the peptide Ac-RRLIEDAE(pY)AARGamide as a model.…”

Section: Ap Maldi/itmsmentioning

confidence: 86%

“…Several groups have investigated the gas phase dephosphorylation patterns of phosphorylated peptides and proteins [8 -21]. DeGnore and Qin [12] studied phosphopeptides by ESI/ ITMS. They reported that the loss of phosphate group is charge state dependent and that loss of phosphate from phosphoserine and phosphothreonine occurs via ␤-elimination.…”

mentioning

confidence: 99%

Fragmentation of phosphopeptides by atmospheric pressure MALDI and ESI/ion trap mass spectrometry

Moyer

Cotter

Woods

2002

An investigation of phosphate loss from phosphopeptide ions was conducted, using both atmospheric pressure matrix-assisted laser desorption/ionization (AP MALDI) and electrospray ionization (ESI) coupled to an ion trap mass spectrometer (ITMS). These experiments were carried out on a number of phosphorylated peptides in order to investigate gas phase dephosphorylation patterns associated with phosphoserine, phosphothreonine, and phosphotyrosine residues. In particular, we explored the fragmentation patterns of phosphotyrosine containing peptides, which experience a loss of 98 Da under collision induced dissociation (CID) conditions in the ITMS. The loss of 98 Da is unexpected for phosphotyrosine, given the structure of its side chain. The fragmentation of phosphoserine and phosphothreonine containing peptides was also investigated. While phosphoserine and phosphothreonine residues undergo a loss of 98 Da under CID conditions regardless of peptide amino acid composition, phosphate loss from phosphotyrosine residues seems to be dependent on the presence of arginine or lysine residues in the peptide sequence. was coupled to an ion trap mass spectrometer (ITMS) [2][3][4][5]. AP MALDI offers the advantages typically associated with a MALDI source such as minimum sample cleanup, ease of sample preparation, multiple analyses from a single spot, as well as simplified spectra for complex mixtures, that are easily interpreted. At the same time, AP MALDI does not require a vacuum region and is easily interchangeable with other atmospheric pressure sources, such as electrospray ionization (ESI). Coupling the AP-MALDI source with an ion trap mass analyzer combines the benefits of MALDI sample preparation and simplicity of spectral analysis resulting from the production of predominantly singly charged ions, with the MS n capabilities of the quadrupole ion trap mass spectrometer [2,3]. This configuration has proven to be useful in obtaining structural information for peptides and protein digests [2,3,6,7] as well as for the identification and characterization of posttranslational modifications [7].Phosphorylation is one of the most common and physiologically important posttranslational modifications in proteins and peptides. Phosphorylation plays a crucial role in a number of biochemical interactions that control the steps necessary for the smooth operation of a normal cell. However, because phosphorylation is an energy consuming process and cells are the most efficient energy consumer in the biological and mechanical world, only a low number of copies (20% on average) of a possible consensus site are usually phosphorylated. To complicate matters, the addition of a negatively charged group to a serine, threonine, or tyrosine residue, which is often surrounded by negatively charged residues such as Asp or Glu (casein kinase consensus sites), often results in suppression of the ion signal in the mass spectra of such peptides.The ubiquitous nature of phosphorylation in biological systems has necessitated the development of method...

“…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

112

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...