Combined electron capture and infrared multiphoton dissociation for multistage MS/MS in a Fourier transform ion cyclotron resonance mass spectrometer

We have mounted a permanent on-axis dispenser cathode electron source inside the magnet bore of a 9.4-T Fourier transform ion cyclotron resonance mass spectrometer. This configuration allows electron capture dissociation (ECD) to be performed reliably on a millisecond time scale. We have also implemented an off-axis laser geometry that enables simultaneous access to ECD and infrared multiphoton dissociation (IRMPD). Optimum performance of both fragmentation techniques is maintained. The analytical utility of performing either ECD or IRMPD on a given precursor ion population is demonstrated by structural characterization of several posttranslationally modified peptides: IRMPD of phosphorylated peptides results in few backbone (b- and y-type) cleavages, and product ion spectra are dominated by neutral loss of H3PO4. In contrast, ECD provides significantly more backbone (c- and z*-type) cleavages without loss of H3PO4. For N-glycosylated tryptic peptides, IRMPD causes extensive cleavage of the glycosidic bonds, providing structural information about the glycans. ECD cleaves all backbone bonds (except the N-terminal side of proline) in a 3-kDa glycopeptide with no saccharide loss. However, only a charge-reduced radical species and some side chain losses are observed following ECD of a 5-kDa glycopeptide from the same protein. An MS3 experiment involving IR laser irradiation of the charge-reduced species formed by electron capture results in extensive dissociation into c- and z-type fragment ions. Mass-selective external ion accumulation is essential for the structural characterization of these low-abundance (modified) peptides.     

Peptide de novo sequencing facilitated by a dual-labeling strategy

A novel peptide derivatization strategy based on guanidination and amidination is presented. Mass-coded labels help distinguish N- and C-terminal fragment ions produced by collision-induced dissociation and are of general utility since peptide N-termini are coded. The amidine labels also promote specific fragmentation pathways that elucidate N-terminal residues and provide valuable internal calibrants. This strategy is demonstrated with the tryptic peptides of several model proteins, including two that are phosphorylated. Additionally, interpreted peptide sequences are matched against a database of over 80,000 proteins to assess the selectivity of this sequencing approach.     

Phosphopeptide/phosphoprotein mapping by electron capture dissociation mass spectrometry

Of methods for dissociation of multiply charged peptide and protein ions, electron capture dissociation (ECD) has the advantages of cleaving between a high proportion of amino acids, without loss of such posttranslational modifications as glycosylation and carboxylation. Here this capability is successfully extended to phosphorylation, for which collisionally activated dissociation (CAD) can cause extensive loss of H3PO4 and HPO3. As shown here, these losses are minimal in ECD spectra, an advantage for measuring the degree of phosphorylation. For phosphorylated peptides, ECD and CAD spectra give complementary backbone cleavages for identifying modification sites. For a 24-kDa heterogeneous phosphoprotein, bovine beta-casein, activated ion ECD cleaved 87 of 208 backbone bonds that identified a phosphorylation site at Ser-15, and localized three more among Ser-17,-18, -19, and -22 and Thr-24, and the last among four other sites. This is the first direct site-specific characterization of this key post-translational modification on a protein without its prior degradation, such as proteolysis.     

Evaluation of front-end higher energy collision-induced dissociation on a benchtop dual-pressure linear ion trap mass spectrometer for shotgun proteomics

We report the implementation of front-end higher energy collision-induced dissociation (fHCD) on a benchtop dual-pressure linear ion trap. Software and hardware modifications were employed, described in detail vide-infra, to allow isolated ions to undergo collisions with ambient gas molecules in an intermediate multipole (q00) of the instrument. Results comparing the performance of fHCD and resonance excitation collision-induced dissociation (RE-CID) in terms of injection time, total number of scans, efficiency, mass measurement accuracy (MMA), unique peptide identifications, and spectral quality of labile modified peptides are presented. fHCD is approximately 23% as efficient as RE-CID, and depending on the search algorithm, it identifies 6.6% more or 15% less peptides (q < 0.01) from a soluble whole-cell lysate ( Caenorhabditis elegans ) than RE-CID using Mascot or Sequest search algorithms, respectively. fHCD offers a clear advantage for the analysis of phosphorylated and glycosylated (O-GlcNAc) peptides as the average cross-correlation score (XCorr) for spectra using fHCD was statistically greater (p < 0.05) than for spectra collected using RE-CID.     

Selective, sensitive, and rapid phosphopeptide identification in enzymatic digests using ESI-FTICR-MS with infrared multiphoton dissociation

Rapid screening for phosphopeptides within complex proteolytic digests involving electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR-MS) in the negative ion mode with infrared multiphoton dissociation (IRMPD) accompanied by improved phosphopeptide sensitivity and selectivity is demonstrated with the tryptic digests of the naturally phosphorylated proteins bovine alpha- and beta-casein. All peptides in a complex proteolytic digest can be examined simultaneously for phosphorylation with a 4-s IR laser pulse at 7-11 W where phosphopeptide signature ions form upon irradiation, as the low energy of activation phosphate moiety cleavage transpires without the dissociation of the unphsophorylated peptide population. The tyrosine phosphorylated peptide HGLDN-pY-R, its nonphosphorylated analogue HGLDNYR, the kinase domain of insulin receptor unphosphorylated TRDIYETDYYRK, monophosphorylated TRDIYED-pY-YRK, and triphosphorylated TRDI-pY-ETD-pY-pY-RK were also used as model peptides in this research. The sensitivity and selectivity of phosphopeptides is shown to greatly improve when the volatile base piperidine is used to adjust the pH of th     

Facile identification of phosphorylation sites in peptides by radical directed dissociation

Identification of phosphorylation sites is of interest due to their importance in protein regulation; however, the identification of the exact sites of this modification is not always easily obtained due to the dynamic nature of phosphorylation and the challenges faced during mass spectrometric analysis. Herein we elaborate on our previous communication (Diedrich, J. K.; Julian, R. R. J. Am. Chem. Soc. 2008, 130, 12212-12213) describing a novel technique for assignment of phosphorylation in a site-specific and facile manner. Phosphorylation sites are selectively modified through β elimination followed by Michael addition chemistry to install a photolabile group. Photodissociation with 266 nm light yields homolytic cleavage at the modification site, generating a β radical which is poised to fragment the peptide backbone. Dissociation primarily yields d-type ions at the previously phosphorylated residue, allowing facile identification. Radical directed fragmentation also occurs in smaller abundances at neighboring residues. The mechanisms behind this selective radical fragmentation are presented and the utility is discussed. Fragmentation is shown to be independent of charge state allowing analysis of a wide variety of peptide sequences including peptides with multiple phosphorylation sites. A comparison of this technique is made with collision induced dissociation (CID) and electron capture dissociation (ECD) for representative peptides.     

Separation and detection of phosphorylated and nonphosphorylated peptides in liquid chromatography-mass spectrometry using monolithic columns and acidic or alkaline mobile phases

Efficient chromatographic separation is a prerequisite for the sensitive analysis of complex peptide mixtures using liquid chromatography-mass spectrometry. This is especially true for the analysis of mixtures of unmodified and posttranslationally modified peptides, for example, phosphorylated peptides in the presence of their unmodified analogues. Applying monolithic capillary columns based on poly(styrene/divinylbenzene), the influence of acidic eluents based on trifluoroacetic and heptafluorobutyric acid as well as an alkaline eluent based on triethylamine-acetic acid (pH 9.2) on the separation of synthetic phosphopeptides was evaluated. Heptafluorobutyric acid offered the longest retention times and highest selectivities and, hence, the most effective separation. Application of the alkaline eluent in conjunction with detection in negative ion mode electrospray ionization mass spectrometry, on the other hand, allowed the detection of phosphorylated peptides with significantly lower limits of detection, as compared to acidic eluents in combination with detection in positive ion mode. Pairs of phosphorylated and nonphosphorylated synthetic peptides, ranging from 7- to 16-mers, as well as phosphorylated peptides form a tryptic protein digest could be separated both at acidic and alkaline pH. Utilizing a 60 x 0.20-mm-i.d. capillary column, the limit of detection in negative ion detection mode for a 4-fold phosphorylated peptide in a beta-casein digest was 10 fmol. Together with the capability for fast separation of protein digests, monolithic columns, thus, facilitate the effective and sensitive analysis of this important posttranslational modification.     

Selective extraction and enrichment of multiphosphorylated peptides using polyarginine-coated diamond nanoparticles

Despite recent advances in phosphopeptide research, detection and characterization of multiply phosphorylated peptides have been a challenge. This work presents a new strategy that not only can effectively extract phosphorylated peptides from complex samples but also can selectively enrich multiphosphorylated peptides for direct matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analysis. Polyarginine-coated diamond nanoparticles are the solid-phase extraction supports used for this purpose. The supports show an exceptionally high affinity for multiphosphorylated peptides due to multiple arginine-phosphate interactions. The efficacy of this method was demonstrated by analyzing a small volume (50 microL) of tryptic digests of proteins such as beta-casein, alpha-casein, and nonfat milk at a concentration as low as 1 x 10 (-9) M. The concentration is markedly lower than that can be achieved by using other currently available technologies. We quantified the enhanced selectivity and detection sensitivity of the method using mixtures composed of mono- and tetraphosphorylated peptide standards. This new affinity-based protocol is expected to find useful applications in characterizing multiple phosphorylation sites on proteins of interest in complex and dilute analytes.     

Selective extraction and characterization of a histidine-phosphorylated peptide using immobilized copper(II) ion affinity chromatography and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Phosphorylation is the predominant posttranslational modification involved in regulating enzymatic activity and mediating signal transduction in prokaryotic and eukaryotic cells. Selective enrichment of phosphorylated peptides prior to mass spectrometric analysis facilitates identification of phosphorylated proteins, determination of specific phosphorylated residues, and characterization of the conditions under which phosphorylation occurs. Such protocols have been established for peptides containing residues that form phosphoesters, such as serine and threonine, using immobilized metal-ion affinity chromatography. Despite the importance of histidine phosphorylation in two-component signal transduction pathways, similar protocols for peptides containing phosphorylated histidine (P-His) residues have proven elusive, due to the instability of these modifications and the propensity of unphosphorylated histidines to interact with immobilized metals ions. We describe a method for the selective extraction of a P-His-containing peptide using immobilized copper(II) ions and disposable metal-chelating pipet tips (ZipTipMC, Millipore). The method is contingent upon pH-dependent interactions between the phosphate group and immobilized copper(II) ions. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry with postsource decay confirms the identity and phosphorylation state of the extracted peptide. Peptides containing unphosphorylated histidine residues or other phosphorylated amino acids are not retained, demonstrating the specificity of the method for P-His-containing peptides.     

Strategy for the identification of sites of phosphorylation in proteins: neutral loss triggered electron capture dissociation

We have previously demonstrated the suitability of data-dependent electron capture dissociation (ECD) for incorporation into proteomic strategies. The ability to directly determine sites of phosphorylation is a major advantage of electron capture dissociation; however, the low stoichiometry associated with phosphorylation means that phosphopeptides are often overlooked in data-dependent ECD analyses. In contrast, collision-induced dissociation (CID) tends to result in loss of the labile phosphate group, often at the expense of sequence fragments. Here, we demonstrate a novel strategy for the characterization of phosphoproteins which exploits the neutral loss feature of CID such that focused ECD of phosphopeptides is achieved. Peptides eluting from a liquid chromatograph are first subjected to CID, and if a neutral loss of 98 Da (corresponding to H3PO4) from the precursor is observed, ECD of that same precursor is performed; i.e., the method comprises neutral loss triggered ECD (NL-ECD-MS/MS). The method was applied to tryptic digests of beta-casein and alpha-casein. For alpha-casein, four sites of phosphorylation were identified with NL-ECD-MS/MS compared with a single site identified by ECD-MS/MS. The method also resulted in ECD of a doubly phosphorylated peptide. A further benefit of the method is that overall protein sequence coverage is improved. Sequence information from nonphosphorylated peptides is obtained as a result of the CID step.     

Infrared multiphoton dissociation for enhanced de novo sequence interpretation of N-terminal sulfonated peptides in a quadrupole ion trap

Infrared multiphoton dissociation (IRMPD) of N-terminal sulfonated peptides improves de novo sequencing capabilities in a quadrupole ion trap mass spectrometer. Not only does IRMPD promote highly efficient dissociation of the N-terminal sulfonated peptides but also the entire series of y ions down to the y(1) fragment may be detected due to alleviation of the low-mass cutoff problem associated with conventional collisional activated dissociation (CAD) methods in a quadrupole ion trap. Commercial de novo sequencing software was applied for the interpretation of CAD and IRMPD MS/MS spectra collected for seven unmodified peptides and the corresponding N-terminal sulfonated species. In most cases, the additional information obtained by N-terminal sulfonation in combination with IRMPD provided significant improvements in sequence identification. The software sequence tag results were combined with a commercial database searching algorithm to interpret sequence information of a tryptic digest on alpha-casein s1. Energy-variable CAD studies confirmed a 30-40% reduction in the critical energies of the N-terminal sulfonated peptides relative to unmodified peptides. This reduction in dissociation energy facilitates IRMPD in a quadrupole ion trap.     

Accurate mass-driven analysis for the characterization of protein phosphorylation. Study of the human Chk2 protein kinase

We describe the data-dependent analysis of protein phosphorylation using rapid-acquisition nano-LC-linear quadrupole ion trap Fourier transform ion cyclotron resonance mass spectrometry (nano-LC-FTMS). The accurate m/z values of singly, doubly, and triply charged species calculated from the theoretical protonated masses of peptides phosphorylated at all Ser, Thr, or Tyr residues of the human checkpoint 2 (Chk2) protein kinase were used for selected ion extraction and chromatographic analysis. Using a kinase-inactive Chk2 mutant as a control, accurate mass measurements from FTMS and collision-induced dissociation spectra, 11 novel Chk2 autophosphorylation sites were assigned. Additionally, the presence of additional Chk2 phosphorylation sites in two unique peptides was deduced from accurate mass measurements. Selected ion chromatograms of all Chk2 phosphopeptides gave single peaks except in three cases in which two closely eluting species were observed. These pairs of phosphopeptides were determined to be positional isomers from MS/MS analysis. In this study, it was also found that ions due to the neutral loss of phosphoric acid from the parent peptide ion were not prominent in 18 of 36 MS/MS spectra of O-linked Chk2 phosphopeptides. Thus, accurate mass-driven analysis and rapid parallel MS/MS acquisition is a useful method for the discovery of new phosphorylation sites that is independent of the signature losses from phosphorylated amino acid residues.     

Ultrasensitive identification of localization variants of modified peptides using ion mobility spectrometry

Localization of the modification sites on peptides is challenging, particularly when multiple modifications or mixtures of localization isomers (variants) are involved. Such variants commonly coelute in liquid chromatography and may be undistinguishable in tandem mass spectrometry (MS/MS) for lack of unique fragments. Here, we have resolved the variants of singly and doubly phosphorylated peptides employing drift tube ion mobility spectrometry (IMS) coupled to time-of-flight mass spectrometry. Even with a moderate IMS resolving power of ～80-100, substantial separation was achieved for both 2+ and 3+ ions normally generated by electrospray ionization, including for the variants indistinguishable by MS/MS. Variants often exhibit a distribution of 3-D conformers, which can be adjusted for optimum IMS separation by prior field heating of ions in a funnel trap. The peak assignments were confirmed using MS/MS after IMS separation, but known species could be identified using just the ion mobility "tag". Avoiding the MS/MS step lowers the detection limit of localization variants to <100 amol, an order of magnitude better than that provided by electron transfer dissociation in an Orbitrap MS.     

Methyl esterification assisted MALDI FTMS characterization of the orcokinin neuropeptide family

Methyl esterification of a peptide converts carboxylic acids, such as those present on the side chains of aspartic (D) and glutamic acid (E) as well as the free carboxyl terminus, to their corresponding methyl esters. This method has been applied to peptide and protein quantitation, de novo sequencing, and reduction of nonspecific binding in immobilized metal affinity chromatography for enrichment of phosphorylated peptides. In this study, we investigate the application of this derivatization reaction to the identification and characterization of the orcokinin neuropeptide family by screening and localizing the acidic side chains in peptides. The methyl esterification reaction drastically improves the fragmentation efficiency of modified orcokinins due to blockage of the aspartate selective cleavage pathway of the native orcokinin peptides. With the improved sustained off-resonance irradiation-collisional-induced dissociation spectra, the number and the locations of D and E residues are easily deduced. In addition, a side reaction that occurs at the carboxamide group of asparagine (N) is studied. The deamidation followed by subsequent methyl esterification reaction mechanism is proposed based on the study of an isotope-labeled standard N*FDEIDR. Reaction kinetics is studied by elevating the temperature from room temperature to 37 degrees C. The deamidation-methyl esterification products are greatly enhanced with elevated reaction temperature. Furthermore, we also explore the utility of this side reaction for rapid screening and characterization of C-terminally amidated neuropeptides. This derivatization reaction is applied to both in situ direct tissue neuropeptide analysis and the analysis of HPLC fractions from the separation of complex neuronal tissue extracts. Overall, this study reports a simple and effective method for profiling and localizing acidic amino acid residues (D/E), amide-containing residues (N/Q), and the C-terminal amide group in a peptide.     

Fragmentation of singly protonated peptides via a combination of infrared and collisional activation

The coupling of matrix-assisted laser desorption/ionization (MALDI) to Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) provides an exceptionally capable platform for peptide analysis, but an important limitation of this approach is the difficulty in obtaining informative tandem mass spectra (MS/MS) of singly protonated peptides. This difficulty is especially pronounced with peptide ions containing basic amino acid residues (for example, tryptic peptides). While such ions can be fragmented in some instrument configurations, most FTICR instruments have comparatively little facility for high-energy fragmentation. Here, a novel MS/MS approach implemented with MALDI-FTICR-MS and specifically intended for enhanced fragmentation of singly protonated peptides is described. The method involves infrared irradiation in concert with the simultaneous application of sustained off-resonance irradiation collision-induced dissociation (SORI-CID). This form of MS/MS, described as a combination of infrared and collisional activation (CIRCA), is shown to provide a greater capacity for dissociation of singly charged model peptide ions as compared to infrared multiphoton dissociation (IRMPD) or SORI-CID alone. Overall, the CIRCA approach is demonstrated to be a feasible technique for accessing useful fragmentation pathways of singly charged peptides, including those harboring basic amino acid residues--a crucial feature in the context of proteomics.     

Enhanced ionization of phosphorylated peptides during MALDI TOF mass spectrometry

Although alpha-cyano-4-hydroxycinnamic acid functions as an excellent matrix for the analysis of most peptides using matrix-assisted laser desorption/ionization time-of-flight (MALDI TOF) mass spectrometry, the ionization of phosphorylated peptides is usually suppressed by nonphosphorylated peptides. As an alternative matrix, 2',4',6'-trihydroxyacetophenone (THAP) with diammonium citrate was found to overcome this problem for the MALDI TOF mass spectrometric analysis of proteolytic digests of phosphorylated proteins. Specifically, the abundances of phosphorylated peptides in tryptic digests of bovine beta-casein and protein kinase C (PKC)-treated mouse cardiac troponin I were enhanced more than 10-fold using THAP during positive ion MALDI TOF mass spectrometry. The protonated molecules of phosphorylated peptides were sufficiently abundant that postsource decay TOF mass spectrometry was used to confirm the number of phosphate groups in each peptide. Finally, tryptic digestion followed by analysis using MALDI TOF mass spectrometry with THAP as the matrix facilitated the identification of a unique phosphorylation site in PKC-treated troponin I.     

Ligand-exchange detection of phosphorylated peptides using liquid chromatography electrospray mass spectrometry

Electrospray ionization mass spectrometry (ESI-MS) is used to selectively detect analytes with a high affinity for metal ions. The detection method is based on the selective monitoring of a competing ligand at its specific m/z value that is released during the ligand-exchange reaction of a metal-ligand complex with analyte(s) eluting from a reversed-phase liquid chromatography column. The ligand-exchange reaction proceeds in a postcolumn reaction detection system placed prior to the inlet of the electrospray MS interface. The feasibility of metal affinity detection by ESI-MS is demonstrated using phosphorylated peptides and iron(III)methylcalcein blue as reactant, as a model system. Methylcalcein blue (MCB) released upon interaction with phosphorylated peptides is detected at m/z 278. The ligand-exchange detection is coupled to a C8 reversed-phase column to separate several nonphosphorylated enkephalins and the phosphorylated peptides pp60 c-src (P) and M2170. Detection limits of 2 microM were obtained for pp60 c-src (P) and M2170. The linearity of the detection method is tested in the range of 2-80 micromol/L phosphorylated compounds (r(2) = 0.9996), and a relative standard deviation of less than 8% (n = 3) for all MCB responses of the different concentrations of phosphorylated compounds was obtained. The presented method showed specificity for phosphorylated peptides and may prove a useful tool for studying other ligand-exchange reactions and metal-protein interactions.     

Mesoporous TiO2-based experimental layout for on-target enrichment and separation of multi- and monophosphorylated peptides prior to analysis with matrix-assisted laser desorption-ionization mass spectrometry

A simple method for on-target enrichment and subsequent separation and analysis of phosphorylated peptides is presented. The tryptic digest of a phosphorylated protein, in this case β-casein, is loaded onto a spot on a thin stripe made of mesoporous TiO(2) sintered onto a conductive glass surface. After washing with a salicylic buffer in order to remove the nonphosphorylated peptides, the stripe is placed in an elution chamber containing a phosphate solution. In a way analogous to thin layer chromatography (TLC), the phosphate solution acts as an eluent, clearly separating multi- and monophosphorylated peptides. By performing matrix-assisted laser desorption-ionization mass spectrometry (MALDI-MS) along the stripe, the detection of all phosphorylated peptides present in the digest is facilitated, as they are isolated from each other. The method was also tested on commercial drinking milk, achieving successful separation between multi- and monophosphorylated peptides, as well as a detection limit in the femtomole range. As the enrichment, separation, and analysis take place in the same substrate, sample handling and risk of contamination and sample loss is minimized. The results obtained suggest that the method, once optimized, may successfully provide a complete phosphoproteome.     

How Peptides Dissociate in Plasmonic Hot Spots

Plasmon‐induced hot carriers enable dissociation of strong chemical bonds by visible light. This unusual chemistry has been demonstrated for several diatomic and small organic molecules. Here, the scope of plasmon‐driven photochemistry is extended to biomolecules and the reactivity of proteins and peptides in plasmonic hot spots is described. Tip‐enhanced Raman spectroscopy (TERS) is used to both drive the reactions and to monitor their products. Peptide backbone bonds are found to dissociate in the hot spot, which is reflected in the disappearance of the amide I band in the TER spectra. The observed fragmentation pathway involves nonthermal activation, presumably by dissociative capture of a plasmon‐induced hot electron. This fragmentation pathway is known from electron transfer dissociation (ETD) of peptides in gas‐phase mass spectrometry (MS), which suggests a general similarity between plasmon‐induced photochemistry and nonergodic reactions triggered by electron capture. This analogy may serve as a design principle for plasmon‐induced reactions of biomolecules.     

Analysis of histidine phosphorylation using tandem MS and ion-electron reactions

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.