MALDI-PSD-MS analysis of the phosphorylation sites of caseinomacropeptide

Talbo, Gert; Suckau, Detlev; Malkoski, Marina; Reynolds, Eric C.

doi:10.1016/s0196-9781(01)00426-0

Cited by 37 publications

(23 citation statements)

References 6 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…For all monophosphorylated isoforms of -casein, the MS/MS spectrum of this peptide allowed the localization of the phosphorylation at the same residue, Ser170. These results are consistent with previous studies, which showed that -casein was fully phosphorylated at Ser170 and only partially at Ser148 (33). Surprisingly, monophosphorylated variant A isoforms appeared at different isoelectric point values on the 2D gel, which could not be explained by differences in the measured molecular masses and by the phosphorylation localization.…”

Section: Discussionsupporting

confidence: 92%

Characterization of Protein Variants and Post-Translational Modifications: ESI-MSn Analyses of Intact Proteins Eluted from Polyacrylamide Gels

Claverol

Burlet‐Schiltz

Gairin

et al. 2003

Molecular & Cellular Proteomics

View full text Add to dashboard Cite

We have developed a strategy to characterize protein isoforms, resulting from single-point mutations and posttranslational modifications. This strategy is based on polyacrylamide gel electrophoresis separation of protein isoforms, mass spectrometry (MS) and MS n analyses of intact proteins, and tandem MS analyses of proteolytic peptides. We extracted protein isoforms from polyacrylamide gels by passive elution using SDS, followed by nanoscale hydrophilic phase chromatography for SDS removal. We performed electrospray ionization MS analyses of the intact proteins to determine their molecular mass, allowing us to draw hypotheses on the nature of the modification. In the case of labile post-translational modifications, like phosphorylations and glycosylations, we conducted electrospray ionization MS n analyses of the intact proteins to confirm their presence. Finally, after digestion of the proteins in solution, we performed tandem MS analyses of the modified peptides to locate the modifications. Using this strategy, we have determined the molecular mass of 5-10 pmol of a protein up to circa 50 kDa loaded on a gel with a 0.01% mass accuracy. The efficiency of this approach for the characterization of protein variants and post-translational modifications is illustrated with the study of a mixture of -casein isoforms, for which we were able to identify the two major variants and their phosphorylation site and glycosylation motif. We believe that this strategy, which combines two-dimensional gel electrophoresis and mass spectrometric analyses of geleluted intact proteins using a benchtop ion trap mass spectrometer, represents a promising approach in proteomics. Molecular & Cellular Proteomics 2:483-493, 2003.The most widely used approach in proteomics using mass spectrometry (MS) 1 is referred to as "bottom up" strategy. In this approach, protein identification is achieved after protein separation by one-or two-dimensional (1D or 2D) polyacrylamide gel electrophoresis followed by protein digestion usually with trypsin, analyses of the resulting peptide mixture using various MS approaches, and data base search. To overcome limitations of 2D gel electrophoresis, alternative techniques based on multidimensional liquid chromatography have been recently developed (1, 2). The "bottom up" strategy has allowed the identification of thousands of proteins in complex mixtures (3). The low protein sequence coverage often obtained using the "bottom up" approach hinders the characterization of post-translational modifications, singlepoint mutations, and truncated forms of a protein. Moreover, the molecular mass of the intact protein is not directly accessible. Recently, "top down" approaches have been described, which are based on mass spectrometric analyses of intact proteins (4). Protein identification is obtained after data base search using the measured protein molecular mass and protein sequence tags determined from MS and tandem MS (MS/MS) analyses of the intact protein, respectively. The critical step in this strategy is t...

show abstract

Section: Discussionsupporting

confidence: 92%

Characterization of Protein Variants and Post-Translational Modifications: ESI-MSn Analyses of Intact Proteins Eluted from Polyacrylamide Gels

Claverol

Burlet‐Schiltz

Gairin

et al. 2003

Molecular & Cellular Proteomics

View full text Add to dashboard Cite

show abstract

“…Currently, mass spectrometry appears to be the most appropriate analytical tool for investigating and characterizing the modified amino acids thanks to its sensitivity, specificity, and rapidity. Different modifications have been studied successfully by MS or MS/MS such as phosphorylation (Talbo et al, 2001;Bennett et al, 2002;Kalume, Molina, & Pandey, 2003;Reinders & Sickmann, 2005), methionine oxidation (Jiang & Smith, 1996), arginine methylation (Boisvert et al, 2003), glycosylation (Kelleher, 2000), N-terminal acetylation (Hesketh et al, 2002), or the formation of disulfide bridges (Jones, Patterson, & Lu, 1998;Jespersen et al, 1999). Clearly, proteins are highly modified and all these modifications complicate their identifications in databases (Nielsen,Savitski,& FIGURE 4.…”

Section: Study Of Covalent Modificationsmentioning

confidence: 99%

“…The main process is called postsource decay (PSD) and corresponds to the fragmentation of the selected monocharged peptide during its flight in the field free drift region in the TOF analyzer (Spengler et al, 1992;Kaufmann, Kirsch, & Spengler, 1994;Kaufmann et al, 1996;Spengler, 1997). PSD mode can give different informative data as sequence and PTMs identification (Chaurand, Luetzenkirchen, & Spengler, 1999;Hoffmann et al, 1999;Talbo et al, 2001;Hardouin et al, 2006). Nevertheless, it is not very often used because of the less intense fragmentation, the large precursor selection width and the time-consuming for spectra acquisition.…”

Section: Introductionmentioning

confidence: 99%

Protein sequence information by matrix‐assisted laser desorption/ionization in‐source decay mass spectrometry

Hardouin

2007

Mass Spectrometry Reviews

137

143

View full text Add to dashboard Cite

Proteins from biological samples are often identified by mass spectrometry (MS) with the two following "bottom-up" approaches: peptide mass fingerprinting or peptide sequence tag. Nevertheless, these strategies are time-consuming (digestion, liquid chromatography step, desalting step), the N- (or C-) terminal information often lacks and post-translational modifications (PTMs) are hardly observed. The in-source decay (ISD) occurring in a matrix assisted laser desorption/ionization (MALDI) source appears an interesting analytical tool to obtain N-terminal sequence, to identify proteins and to characterize PTMs by a "top-down" strategy. The goal of this review deals with the usefulness of the ISD technique in MALDI source in proteomics fields. In the first part, the ISD principle is explained and in the second part, the use of ISD in proteomic studies is discussed for protein identification and sequence characterization.

show abstract

“…It comprises the 106 to 169 C-terminal fragment of -casein and contains all the posttranslational modification sites found in -casein. CMP is both variably phosphorylated and glycosylated (23,25,31). CMP is completely phosphorylated at Ser 149 and partially phosphorylated (Ϸ10%) at Ser 127 as determined by matrix-assisted laser desorption ionization-time of flight (MALDI)-post source delay (PSD) mass spectrometry (31).…”

mentioning

confidence: 99%

“…CMP is both variably phosphorylated and glycosylated (23,25,31). CMP is completely phosphorylated at Ser 149 and partially phosphorylated (Ϸ10%) at Ser 127 as determined by matrix-assisted laser desorption ionization-time of flight (MALDI)-post source delay (PSD) mass spectrometry (31). Additionally, there are at least six genetic variants of -casein, with variants A and B being by far the most common (5 (15).…”

mentioning

confidence: 99%

Divalent Metal Cations Increase the Activity of the Antimicrobial Peptide Kappacin

Dashper

O'Brien-Simpson

Cross

et al. 2005

Antimicrob Agents Chemother

Self Cite

View full text Add to dashboard Cite

Kappacin, nonglycosylated -casein(106-169), is a novel antimicrobial peptide produced from -casein found in bovine milk. There are two major genetic forms of kappacin, A and B, and using synthetic peptides corresponding to the active region, -casein(138-158), of these forms, we have shown that the Asp 148 to Ala 148 substitution is responsible for the lesser antibacterial activity of -casein-B(106-169). Kappacin was shown to have membranolytic action at concentrations above 30 M at acidic pH when tested against artificial liposomes. There was little membranolytic activity at neutral pH, which is consistent with the lack of antibacterial activity of kappacin against Streptococcus mutans at this pH. Kappacin specifically bound two zinc or calcium ions per mol, and this binding enhanced antibacterial activity at neutral pH. Nuclear magnetic resonance analysis indicated that a -casein-A(138-158) synthetic peptide undergoes a conformational change in the presence of the membrane solvent trifluoroethanol and excess divalent metal ions. This change in conformation is presumably responsible for the increase in antibacterial activity of kappacin detected in the presence of excess zinc or calcium ions at neutral pH. When tested against the oral bacterial pathogen S. mutans cultured as a biofilm in a constant-depth film fermentor, a preparation of 10 g/liter kappacin and 20 mM ZnCl 2 reduced bacterial viability by 3 log 10 and suppressed recovery of viability. In contrast 20 mM ZnCl 2 alone reduced bacterial viability by Ϸ1 log 10 followed by rapid recovery. In conclusion, kappacin has a membranolytic, antibacterial effect that is enhanced by the presence of divalent cations.

show abstract

MALDI-PSD-MS analysis of the phosphorylation sites of caseinomacropeptide

Cited by 37 publications

References 6 publications

Characterization of Protein Variants and Post-Translational Modifications: ESI-MSn Analyses of Intact Proteins Eluted from Polyacrylamide Gels

Characterization of Protein Variants and Post-Translational Modifications: ESI-MSn Analyses of Intact Proteins Eluted from Polyacrylamide Gels

Protein sequence information by matrix‐assisted laser desorption/ionization in‐source decay mass spectrometry

Divalent Metal Cations Increase the Activity of the Antimicrobial Peptide Kappacin

Contact Info

Product

Resources

About