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The application of electron-transfer dissociation (ETD) to obtain single-residue resolution in hydrogen exchange-mass spectrometry (HX-MS) experiments has recently been demonstrated. For such measurements, it is critical to ensure that the level of gas-phase hydrogen scrambling is negligible. Here we utilize the abundant loss of ammonia upon ETD of peptide ions as a universal reporter of positional randomization of the exchangeable hydrogens (hydrogen scrambling) during HX-ETD experiments. We show that the loss of ammonia from peptide ions proceeds without depletion of deuterium when employing optimized mild electrospray ion source settings for the HX-ETD analysis of a selectively labeled model peptide and peptides derived from fully labeled [beta]-microglobulin. Hydrogen scrambling, as induced by excessive vibrational excitation of peptide ions during harsh declustering conditions, is easily detected by a depletion of deuterium when deuterated ammonia is lost from peptides during ETD. This straightforward method requires no modifications to the experimental workflow and has the great advantage that the occurrence of hydrogen scrambling can be directly detected in the actual peptides analyzed in the HX-ETD experiment. 10.1021/ac101889b

Because of unparalleled sensitivity and tolerance to protein size, mass spectrometry (MS) has become a popular method for measuring the solution hydrogen ([sup.1]H/[sup.2]H) exchange (HX) of biologically relevant protein states. While incorporated deuterium can be localized to different regions by pepsin proteolysis of the labeled protein, the assignment of deuteriums to individual residues is typically not obtained, thereby limiting a detailed understanding of HX and the dynamics of protein structure. Here we use gas-phase fragmentation of peptic peptides by electron transfer dissociation (ETD) to measure the HX of individual amide linkages in the amyloidogenic protein [[beta].sub.2]-microglobulin. A comparison of the deuterium levels of 60 individual backbone amides of [[beta].sub.2]-microglobulin measured by HX-ETD-MS analysis to the corresponding values measured by NMR spectroscopy shows an excellent correlation. The deuterium labeling pattern of [[beta].sub.2]microglobulin is retained in the gaseous fragment ions by employing mild declustering conditions for electrospray ionization. A recently developed model peptide is used to arrive at such ion source declustering conditions that prevent the occurrence of intramolecular gas-phase hydrogen ([sup.1]H/[sup.2]H) migration (i.e., hydrogen scrambling). This article demonstrates that ETD can be implemented in a mass spectrometric method to monitor the conformational dynamics of proteins in solution at single-residue resolution.

Tandem mass spectrometry (MS/MS) is a powerful tool for characterization of post-translationally modified proteins, including [mamber of]-N-acetyllysine-containing species. Previous reports indicate that [member of]-N-acetyllysine immonium ions are useful marker ions for peptides containing [member of]-Nacetyllysine, but the specificity and sensitivity of these ions for assignment of lysine acetylation by MS/MS have not been studied in detail. We investigated MS/MS data sets of 172 [member of]-N-acetyllysine tryptic peptides and 268 nonacetylated tryptic peptides to establish the utility and reliability of [member of]-N-acetyllysine immonium ions for identification and validation of acetylated peptides. Our analysis shows that the immonium ion at m/z 143 lacks specificity for lysine-acetylated peptides, whereas the derivative at m/z 126 is highly specific (98.1%). We also studied the positional effect of the [member of]-N-acetyllysine on the intensity of observed acetyllysine immonium ions. We observed an increase in acetyllysine immonium ion intensities when the acetylated lysine was N-terminally positioned in the peptide as compared to internal positions. Based on these observations we propose a validation scheme for unambiguous assignment of acetyllysine-containing peptides by MS/MS. Our analysis of [member of]-N-acetyllysine immonium ions provide a framework for investigation of MS/MS marker ion specificity and sensitivity that can be applied in studies of other types of post-translational modifications.

We have tested the effect of m-nitrobenzyl alcohol (m-NBA) as a method to increase the average charge state of protonated gas-phase molecular ions generated by ESI from tryptic peptides and phosphopeptides. Various concentrations of m-NBA were added to the mobile phases of a liquid chromatography system coupled to an ESI tandem mass spectrometer. Addition of just 0.1% m-NBA changed the average charge state for the identified tryptic BSA peptides from 2.2+ to 2.6+. As a result, the predominant charge states for BSA peptides were changed from 2+ to [greater than or equal to] 3+. To evaluate the benefits of peptide charge enhancement, the ETD fragmentation efficiency and Mascot peptide score were compared for BSA peptides in charge states 2+ and 3+. In all cases but one, triply charged peptides fragmented more efficiently than the analogues 2+ peptide ions. On average, triply charged peptides received a 68% higher Mascot score (24 units) than doubly charged peptides, m-NBA also increased the average charge state of phosphopeptides by up to 0.5 charge unit. The ease of implementation and the analytical benefits of charge enhancement of tryptic peptides by addition of m-NBA to the LC solvents suggest the general application of this reagent in proteomic studies that employ ETD-MS/MS and related techniques.

Phosphorylation of proteins is essential in intracellular signal transduction pathways in eukaryotic and prokaryotic cells. Histidine phosphorylation plays an important role in two-component signal transduction in bacteria. In this study, we describe the characterization of a synthetic histidine-phosphorylated peptide with four different mass spectrometric (MS) fragmentation techniques: Collision-induced dissociation (CID), electron capture dissociation, electron-transfer dissociation, and electron detachment dissociation. Furthermore, LC-MS methods were developed to detect histidine-phosphorylated peptides, which are acid-labile, in more complex samples. From these results, we concluded that nonacidic solvent systems or fast LC methods provide the best conditions for separation of histidine-phosphorylated peptides prior to electrospray ionization mass spectrometry analysis. Electron-based fragmentation methods should be used for determination of histidine phosphorylation sites, since CID results in very facile phosphate-related neutral losses. The developed LC-MS/MS methods were successfully applied to a tryptic digest of the cytoplasmic part of the histidine kinase EnvZ, which was in vitro autophosphorylated. Finally, a new method is described for nonretentive solid-phase extraction of histidine-phosphorylated peptides using polymeric Strata-X microcolumns.

Glycosylphosphatidylinositol-anchored proteins (GPI-APs) are posttranslationally processed proteins that become tethered to the extracellular leaflet of the plasma membrane via a C-terminal glycan-like moiety. Since the first GPI-AP was described in the 1970s, more than 500 GPI-APs have been reported in a range of species, including plants, microbes, and mammals. GPI-APs are probably involved in cell signaling, cell recognition, and cell remodeling processes, and they may potentially serve as cell surface antigens or vaccine targets in pathogenic microorganisms or transformed mammalian cells. Due to the structural complexity and physicochemical properties of GPI-APs, their identification and structural characterization is a demanding analytical task. Here, we report a simple, fast and sensitive method for isolation and structural analysis of GPI-anchors using a combination of hydrophilic interaction liquid chromatography and matrix-assisted laser desorption/ionization (MALDI) quadrupole time-of-flight tandem mass spectrometry. This method allowed analysis of GPI peptides derived from low picomole levels of the porcine kidney membrane dipeptidase. Furthermore, it allowed unambiguous assignment of the omega site via amino acid sequencing of the modified peptides. GPI-anchor-specific diagnostic ions were observed by MALDI-MS/MS at m/z 162, 286, 422, and 447, corresponding to glucosamine, mannose ethanolamine phosphate, glucosamine inositol phosphate, and mannose ethanolamine phosphate glucosamine, respectively. Thus, the methodology described herein may enable sensitive and specific detection of GPI-anchored peptides in large-scale proteomic studies of plasma membrane proteins.

Peptide mass mapping by matrix-assisted laser desorption/ionization (MALDI) followed by database searching with the set of measured peptide masses is now a powerful method for the identification of pure proteins. Protein mixtures - such as frequently occur due to comigration in polyacrylamide gel bands - have hitherto required protein sequencing. Here we demonstrate that such protein bands can also be analyzed by peptide mass mapping alone. Database searching with the complete list of peptide masses determined by delayed-extraction MALDI mass spectrometry with a mass error of less than 30 ppm retrieves the most prominent protein in a mixture. In a second step, the protein identity is further confirmed by matching as many of the measured peptide masses as possible to the retrieved amino acid sequence. Peptide masses remaining after this 'second pass search' are searched again to identify the next component in the protein mixture. This iterative process is repeated until all major ion signals are accounted for. Protein mixtures consisting of two or more individual components in a single gel band can be analyzed, further increasing the general applicability of MALDI peptide mapping for protein identification.

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry is a sensitive and versatile method for biomolecular analysis which has potential for high-throughput screening in many applications. To obtain mass spectra of optimal quality, however, laser fluence is continuously adjusted during data acquisition to be close to the threshold level of ion production, requiring a skilled operator and several minutes of acquisition time per sample. Using real-time fuzzy logic control of the laser fluence, we here demonstrate that the acquisition of MALDI spectra can be automated without reduction of data quality. The control algorithm evaluates signal intensity and mass resolution of the base peak. It then regulates the laser fluence to keep the ion signal intensity within the dynamic range of the data acquisition hardware while maintaining high mass resolution. This fuzz logic control system allows unattended data acquisition using either static ion extraction or delayed ion extraction MALDI. Even for difficult samples such as femtomole-level peptide mixtures, no significant reduction in data quality is observed, as compared to manually obtained spectra. Automated analysis of 78 chromatographic fractions with high mass accuracy demonstrates the utility of the method. The control algorithm has been combined with other software modules to completely automate database identification of proteins by their peptide mass maps. The success of fuzzy logic in MALDI automation suggests wider uses of this technique in mass spectrometry.

Tandem mass spectrometry (MS/MS) is a powerful tool for characterization of post-translationally modified proteins, including ε-N-acetyllysine-containing species. Previous reports indicate that ε-N-acetyllysine immonium ions are useful marker ions for peptides containing ε-N-acetyllysine, but the specificity and sensitivity of these ions for assignment of lysine acetylation by MS/MS have not been studied in detail. We investigated MS/MS data sets of 172 ε-N-acetyllysine tryptic peptides and 268 non- acetylated tryptic peptides to establish the utility and reliability of ε-N-acetyllysine immonium ions for identification and validation of acetylated peptides. Our analysis shows that the immonium ion at m/z 143 lacks specificity for lysine-acetylated peptides, whereas the derivative at m/z 126 is highly specific (98.1%). We also studied the positional effect of the ε-N-acetyllysine on the intensity of observed acetyllysine immonium ions. We observed an increase in acetyllysine immonium ion intensities when the acetylated lysine was N-terminally positioned in the peptide as compared to internal positions. Based on these observations we propose a validation scheme for unambiguous assignment of acetyllysine-containing peptides by MS/MS. Our analysis of ε-N-acetyllysine immonium ions provide a framework for investigation of MS/MS marker ion specificity and sensitivity that can be applied in studies of other types of post-translational modifications. [ABSTRACT FROM AUTHOR]