Phosphopeptide analysis by positive and negative ion matrix‐assisted laser desorption/ionization mass spectrometry

Janek, Katharina; Wenschuh, Holger; Bienert, Michael; Krause, Eberhard

doi:10.1002/rcm.417

Cited by 118 publications

(130 citation statements)

References 19 publications

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“…In addition, threonine phosphorylation leads to increase in the ionization efficiency. This result is consistent with the previous MALDI study [9] and is in line with the fact that a phosphorylated side chain can deprotonate readily to render the negatively charged peptide, thereby increasing the ionization efficiency. In this respect, the relative ionization efficiencies were measured following the same procedures as described above for the positive-ion mode.…”

Section: Resultssupporting

confidence: 93%

“…In this method, after normalizing against the signal of suitable proteolytic peptides, the extent of phosphorylation could be determined. In addition, methods for direct measurement of the ionization efficiency changes caused by PTM on peptides were reported [1,8,9]. In these prior studies, the synthetic peptides were first quantified by weight [8] or by amino acid analysis [1] and, after comparing the LC-MS results with known quantification data, the variation in ionization efficiency caused by certain PTMs could be measured.…”

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confidence: 99%

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A method to determine the ionization efficiency change of peptides caused by phosphorylation

Gao

Wang

2007

J. Am. Soc. Mass Spectrom.

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Quantitative assessment of post-translational modifications in proteins by mass spectrometry often requires the consideration of the alteration in ionization efficiency of peptides induced by the modification. Herein, we introduced a method to measure the relative ionization efficiencies of peptides using specifically designed unlabeled peptides. In our design, the peptide under study, in either the unmodified or modified form, is linked with an internal standard peptide via an enzyme cleavage site; thus, after enzymatic digestion, we could obtain readily a 1:1 ratio between the peptide under investigation and the internal standard peptide. The relative ionization efficiencies of the modified and unmodified peptides can then be calculated from the modification-induced change in the ratio of relative abundances of the ion of the peptide of interest over that of the internal standard peptide. We demonstrated the usefulness of the method by assessing the change in ionization efficiencies of four peptides introduced by phosphorylation. (J Am Soc Mass Spectrom 2007, 18, 1973-1976 Methods have been developed to achieve unbiased PTM analysis by using mass spectrometry [2]. In this respect, an isotope-labeled internal standard was used to achieve absolute quantification of PTMs in proteins. For instance, isotope-coded affinity tag (ICAT) [3], stable isotope labeling with amino acids in cell culture (SILAC) [4], and introducing 18 O labeling by enzymatic digestion in H 2 18 O [5] have been employed. In addition, an isotope-dilution method, where the absolute or relative quantification of PTM levels of peptides is achieved by doping a known amount of the synthetic isotope-labeled peptides into the peptide samples as the internal standard, was reported [6].In recent years, an isotope-free method for the quantification of protein phosphorylation was also developed [7]. In this method, after normalizing against the signal of suitable proteolytic peptides, the extent of phosphorylation could be determined. In addition, methods for direct measurement of the ionization efficiency changes caused by PTM on peptides were reported [1,8,9]. In these prior studies, the synthetic peptides were first quantified by weight [8] or by amino acid analysis [1] and, after comparing the LC-MS results with known quantification data, the variation in ionization efficiency caused by certain PTMs could be measured.Herein, we developed a simple isotope-free method to measure the change in ionization efficiency introduced by PTMs, and we demonstrated that the method allowed for the measurement of the alteration in ionization efficiency introduced by phosphorylation of threonine residues in peptides. In our method, the target peptide, in the unmodified or phosphorylated form, is fused with a standard peptide through the recognition site of a proteolytic enzyme (Figure 1). After digestion with the protease, the standard peptide and the peptide under study can be released at a molar ratio of 1:1. The change in ionization efficiency can then be gauged f...

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Section: Resultssupporting

confidence: 93%

mentioning

confidence: 99%

A method to determine the ionization efficiency change of peptides caused by phosphorylation

Gao

Wang

2007

J. Am. Soc. Mass Spectrom.

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“…Our data found that in the presence of p38 MAPK, the 21-aa C-terminal MRP-14 tryptic peptide that contains Thr 113 showed an 80-Da increase in mass, as compared with MRP-14 tryptic peptides in the absence of p38 MAPK. This is consistent with phosphorylation of this peptide, as the mass of a phosphoryl group (HPO 3 ) is 80 Da (40). Use of tandem mass spectrometry allowed us to observe the ␤-elimination, neutral loss of 98 m/z from the precursor peptide ion, which is the mass of phosphoric acid (H 3 PO 4 ) (40,41).…”

Section: Discussionsupporting

confidence: 73%

“…These results indicate that MRP-14 translocates to Triton X-100 insoluble structures at the base of lamellipodia in human neutrophils upon fMLP treatment and that the translocation is p38 MAPK dependent. the 80-Da mass addition of a phosphoryl group (HPO 3 ) (40). Analysis of peptide mass spectra showed a peak at 2177.3 m/z in samples from p38 MAPK treated and untreated MRP-14 (Fig.…”

Section: P38 Mapk Phosphorylates Thr 113 On Mrp-14mentioning

confidence: 98%

Myeloid-Related Protein-14 Is a p38 MAPK Substrate in Human Neutrophils

Lominadze

Rane

Merchant

et al. 2005

The Journal of Immunology

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The targets of the p38 MAPK pathway that mediate neutrophil functional responses are largely unknown. To identify p38 MAPK targets, a proteomic approach was applied in which recombinant active p38 MAPK and [32P]ATP were added to lysates from unstimulated human neutrophils. Proteins were separated by two-dimensional gel electrophoresis, and phosphoproteins were visualized by autoradiography and identified by MALDI-TOF. Myeloid-related protein-14 (MRP-14) was identified as a candidate p38 MAPK substrate. MRP-14 phosphorylation by p38 MAPK was confirmed by an in vitro kinase reaction using purified MRP-14/MRP-8 complexes. The site of MRP-14 phosphorylation by p38 MAPK was identified by tandem mass spectrometry and site-directed mutagenesis to be Thr113. MRP-14 phosphorylation by p38 MAPK in intact neutrophils was confirmed by [32P]orthophosphate loading, followed by fMLP stimulation in the presence and absence of a p38 MAPK inhibitor, SB203580. Confocal microscopy of Triton X-100 permeabilized neutrophils showed that a small amount of MRP-14 was associated with cortical F-actin in unstimulated cells. fMLP stimulation resulted in a p38 MAPK-dependent increase in MRP-14 staining at the base of lamellipodia. By immunoblot analysis, MRP-14 was present in plasma membrane/secretory vesicle fractions and gelatinase and specific granules, but not in azurophil granules. The amount of MRP-14 associated with plasma membrane/secretory vesicle and gelatinase granule fractions increased after fMLP stimulation in a p38 MAPK-dependent manner. Direct phosphorylation of the MRP-14/MRP-8 complex by p38 MAPK increased actin binding in vitro by 2-fold. These results indicate that MRP-14 is a potential mediator of p38 MAPK-dependent functional responses in human neutrophils.

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“…Major drawbacks of these methods are decreased ionization rates of phosphopeptides in complex mixtures due to suppression effects. These effects can be reduced by measuring phosphopeptides in negative ion mode as their ionization is better than in positive mode [55]. Unfortunately, negative-polarity MS/MSspectra are mostly of very poor quality.…”

Section: Localization Of Phosphorylation Sitesmentioning

confidence: 99%

State-of-the-art in phosphoproteomics

2005

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Presently, phosphorylation of proteins is the most studied and best understood PTM. However, the analysis of phosphoproteins and phosphopeptides is still one of the most challenging tasks in contemporary proteome research. Since not every phosphoprotein is accessible by a certain method and identification of the phosphorylated amino acid residue is required in the majority of cases, various strategies for the detection and localization of phosphorylations have been developed. Identification and localization of protein phosphorylations is mostly done by MS nowadays but phosphoproteins and -peptides are often suppressed in comparison to the unphosphorylated species if measured in complex mixtures. Thus, the isolation of pure phosphopeptide samples is a main task. This review gives an overview over the most frequently used methods in isolation and detection of phosphoproteins and -peptides such as specific enrichment or separation strategies as well as the localization of the phosphorylated residues by various mass spectrometric techniques.

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Phosphopeptide analysis by positive and negative ion matrix‐assisted laser desorption/ionization mass spectrometry

Cited by 118 publications

References 19 publications

A method to determine the ionization efficiency change of peptides caused by phosphorylation

A method to determine the ionization efficiency change of peptides caused by phosphorylation

Myeloid-Related Protein-14 Is a p38 MAPK Substrate in Human Neutrophils

State-of-the-art in phosphoproteomics

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