Interrogation of N-Linked oligosaccharides using infrared multiphoton dissociation in FT-ICR mass spectrometry

The structural elucidation of oligosaccharides remains a major challenge. Mass spectrometry provides a rapid and convenient method for structural elucidation based on tandem mass spectrometry. Ions commonly are selected and subjected to collision-induced dissociation (CID) to obtain structural information. Unfortunately, N-linked oligosaccharides are relatively large compounds and are not readily fragmented using CID. In this report, we illustrate the use of infrared multiphoton dissociation (IRMPD) to obtain structural information for large N-linked oligosaccharides. The IRMPD and CID behavior of oligosaccharides were compared for high-mannose-type oligosaccharides. Fragmentation that could not be obtained through conventional CID in Fourier transform ion cyclotron resonance mass spectrometry was observed with N-linked oligosaccharides. O-Linked and N-linked glycans of similarly large sizes were compared. It was found that internal cross-ring cleavages were observed only for N-linked oligosaccharides. The mannose branch points of N-linked oligosaccharides are apparently more susceptible to cross-ring cleavages.     

Enhanced detection and identification of glycopeptides in negative ion mode mass spectrometry

A combined mass spectrometry (MS) and tandem mass spectrometry (MS/MS) approach implemented with matrix-assisted laser desorption ionization Fourier transform ion cyclotron resonance mass spectrometry (MALDI FTICR MS) in the negative ion mode is described for enhanced glycopeptide detection and MS/MS analysis. Positive ion mode MS analysis is widely used for glycopeptide characterization, but the analyses are hampered by potential charge-induced fragmentation of the glycopeptides and poor detection of the glycopeptides harboring sialic acids. Furthermore, tandem MS analysis (MS/MS) via collision-induced dissociation (CID) of glycopeptides in the positive ion mode predominantly yields glycan fragmentation with minimal information to verify the connecting peptide moiety. In this study, glycoproteins such as, bovine lactoferrin (b-LF) for N-glycosylation and kappa casein (k-CN) for O-glycosylation were analyzed in both the positive- and negative ion modes after digestion with bead-immobilized Pronase. For the b-LF analysis, 44 potential N-linked glycopeptides were detected in the positive ion mode while 61 potential N-linked glycopeptides were detected in the negative ion mode. By the same token, more O-linked glycopeptides mainly harboring sialic acids from k-CN were detected in the negative ion mode. The enhanced glycopeptide detection allowed improved site-specific analysis of protein glycosylation and superior to positive ion mode detection. Overall, the negative ion mode approach is aimed toward enhanced N- and O-linked glycopeptide detection and to serve as a complementary tool to positive ion mode MS/MS analysis.     

Analysis of glycopeptides using lectin affinity chromatography with MALDI-TOF mass spectrometry

Glycopeptides prepared from 1 nmol of a mixture of glycoproteins, transferrin, and ribonuclease B by lysylendopeptidase digestion were isolated by lectin and cellulose column chromatographies, and then they were analyzed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry and MALDI-quadrupole ion trap (QIT)-TOF mass spectrometry which enables the performance of MS ( n ) analysis. The lectin affinity preparation of glycopeptides with Sambucus nigra agglutinin and concanavalin A provides the glycan structure outlines for the sialyl linkage and the core structure of N-glycans. Such structural estimation was confirmed by MALDI-TOF MS and MALDI-QIT-TOF MS/MS. Amino acid sequences and location of glycosylation sites were determined by MALDI-QIT-TOF MS/MS/MS. Taken together, the combination of lectin column chromatography, MALDI-TOF MS, and MALDI-QIT-TOF MS ( n ) provides an easy way for the structural estimation of glycans and the rapid analysis of glycoproteomics.     

Hydrophilic affinity isolation and MALDI multiple-stage tandem mass spectrometry of glycopeptides for glycoproteomics

In glycoproteomics, key structural issues, protein identification, locations of glycosylation sites, and evaluation of the glycosylation site microheterogeneity should be easily evaluated in a large number of glycoproteins, while mass spectrometry (MS) provides substantial information about individual purified glycoproteins. Considering that structural issues are elucidated by studying glycopeptides and that the tandem MS of a tryptic peptide composed of several amino acid residues is enough for protein identification, construction of an MS-based method handling tryptic glycopeptides would be of considerable benefit in research. To this end, a simple and efficient method, utilizing hydrophilic binding of carbohydrate matrixes such as cellulose and Sepharose to oligosaccharides, was successfully applied to the isolation of tryptic glycopeptides. Both peptide and oligosaccharide structures were elucidated by multiple-stage tandem MS (MS(n)) of the ions generated by matrix-assisted laser desorption/ionization (MALDI), as follows. The MALDI ion trap mass spectrum of a tryptic glycopeptide mixture from N-linked glycoproteins was composed of the [M + H]+ ions of component glycopeptides. Collision-induced dissociation (CID) of the glycopeptide [M + H]+ ion generated saccharide-spaced peaks, with an interval of, for example, 146, 162, and 203 Da, and their fragment ions corresponding to the peptide and peptide + N-acetylglucosamine (GlcNAc) species in the MS2 spectrum. The saccharide-spaced ladder served to outline oligosaccharide structures, which were then selected as precursors for subsequent MS(n) analyses. The peptide or peptide + GlcNAc ions in the MS2 spectrum or the corresponding ions abundant in the MS1 spectrum were subjected to CID for determination of peptide sequences, to identify proteins and their glycosylation sites. The strategy, isolation of glycopeptides followed by MS(n) analysis, efficiently characterized the structures of beta2-glycoprotein I with four N-glycosylation sites and was applied to an analysis of total serum glycoproteins.     

Tandem parallel fragmentation of peptides for mass spectrometry

Parallel fragmentations of peptides in the source region and in the collision cell of tandem mass spectrometers are sequentially combined to develop parallel collision-induced-dissociation mass spectrometry (p2CID MS). Compared to MS/MS spectra, the p2CID mass spectra show increased signal intensities (2-400-fold) and number of sequence ions. This improvement is attributed to the fact that p2CID MS virtually samples all the ions generated by electrospray ionization, including intact and fragment ions of different charge states from a peptide. We implement the method using a quadrupole time-of-flight tandem mass spectrometer. The instrument is operated in TOF-MS mode that allows the ions from source region broadband-passing the first mass analyzer to enter the collision cell. Cone voltage and collision energy are investigated to optimize the outcome of the two parallel CID processes. In the in-source parallel CID, elevated cone voltage produces singly charged intact peptide ions and large fragment ions, as well as decreases the charge-state distribution of peptide ions mainly to double and single charges. The in-collision-cell parallel CID is optimized to dissociate the ions from the source region to produce small and medium fragment ions. The method of p2CID MS is especially useful for sequencing of large peptides with labile amide bonds and peptides with C-terminal arginine. It has unique potential for de novo sequencing of peptides and proteome analysis, especially for affinity-enriched subproteomes.     

Impact of different ionization methods on the molecular assignments of asphaltenes by FT-ICR mass spectrometry

Over the years, ultrahigh resolution mass spectrometry has successfully illustrated the extreme complexity of crude oil and related solubility or polarity based fractions on a molecular level. However, the applied ionization technique greatly influences the outcome and may provide misleading information. In this work, we investigate the atmospheric pressure laser ionization (APLI) technique coupled with Fourier transform ion cyclotron resonance mass spectrometer to analyze the asphaltene fraction of a crude oil. These results were compared to data obtained by using other existing atmospheric pressure ionization methods. Furthermore elemental analysis and solid state NMR were used to obtain the bulk characteristics of the asphaltene sample. The results of the different ionization techniques were compared with the bulk properties in order to describe the potential discrimination effects of the ionization techniques that were observed. The results showed that APLI expands the range of the assigned molecules, while retaining information already observed with the generally used ion sources.     

Characterization of hydrophobic peptides by atmospheric pressure photoionization-mass spectrometry and tandem mass spectrometry

The use of photoionization at atmospheric pressure shows great potential for the mass analysis of large apolar or hydrophobic peptides. Mass spectra that were obtained using this technique showed mainly singly charged ions. While polar peptides spectra do not produce fragment ions, others lead to B-type or C-type in-source fragmentation. These dissociation reactions, which could involve electron capture dissociation processes in the case of the C-type ions, are observed for hydrophobic peptides. Both the compatibility of this ionization mode with reversed- or normal-phase liquid chromatographic separation and its sensitivity allow liquid chromatography coupling to both mass spectrometry and tandem mass spectrometry for the analyses of hydrophobic peptide mixtures. Atmospheric pressure photoionization seems to be an interesting alternative method to study hydrophobic peptides that are not easily ionizable by more classical ionization techniques such as electrospray ionization and matrix-assisted laser desorption/ionization.     

Ion-molecule reactions for the characterization of polyols and polyol mixtures by ESI/FT-ICR mass spectrometry

A mass spectrometric method is described for the identification and counting of hydroxyl groups in an analyte. Analytes introduced into a FT-ICR mass spectrometer and ionized by positive mode ESI were allowed to react with the neutral reagent diethylmethoxyborane. This results in derivatization of the hydroxyl groups of the analytes by replacement of a proton with a diethylborenium ion. Protonated polyols react by consecutive derivatization reactions, wherein all, or nearly all, of the hydroxyls are derivatized. The polyol derivatization products are separated by 68 mass units in the mass spectrum. This 68 Da mass shift, along with 30 Da mass shifts arising from intramolecular derivatization of the primary derivatization products, makes it easy to count the number of functional groups present in the analyte. The utility of this method for the analysis of polyols as single-component solutions, as mixtures, or in HPLC effluent (LC-MS analysis) is demonstrated.     

In-spray supercharging of peptides and proteins in electrospray ionization mass spectrometry

Enhanced charging, or supercharging, of analytes in electrospray ionization mass spectrometry (ESI MS) facilitates high resolution MS by reducing an ion mass-to-charge (m/z) ratio, increasing tandem mass spectrometry (MS/MS) efficiency. ESI MS supercharging is usually achieved by adding a supercharging reagent to the electrospray solution. Addition of these supercharging reagents to the mobile phase in liquid chromatography (LC)-MS/MS increases the average charge of enzymatically derived peptides and improves peptide and protein identification in large-scale bottom-up proteomics applications but disrupts chromatographic separation. Here, we demonstrate the average charge state of selected peptides and proteins increases by introducing the supercharging reagents directly into the ESI Taylor cone (in-spray supercharging) using a dual-sprayer ESI microchip. The results are comparable to those obtained by the addition of supercharging reagents directly into the analyte solution or LC mobile phase. Therefore, supercharging reaction can be accomplished on a time-scale of ion liberation from a droplet in the ESI ion source.     

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.     

Two-dimensional gas-phase separations coupled to mass spectrometry for analysis of complex mixtures

Ion mobility spectrometry (IMS) has been explored for decades, and its versatility in separation and identification of gas-phase ions is well established. Recently, field asymmetric waveform IMS (FAIMS) has been gaining acceptance in similar applications. Coupled to mass spectrometry (MS), both IMS and FAIMS have shown the potential for broad utility in proteomics and other biological analyses. A major attraction of these separations is extremely high speed, exceeding that of condensed-phase alternatives by orders of magnitude. However, modest separation peak capacities have limited the utility of FAIMS and IMS for analyses of complex mixtures. We report 2-D gas-phase separations that join FAIMS to IMS, in conjunction with high-resolution and accuracy time-of-flight (TOF) MS. Implementation of FAIMS/IMS and IMS/MS interfaces using electrodynamic ion funnels greatly improves sensitivity. Evaluation of FAIMS/IMS/TOF performance for a protein mixture tryptic digest reveals high orthogonality between FAIMS and IMS dimensions and, hence, the benefit of FAIMS filtering prior to IMS/MS. The effective peak capacities in analyses of tryptic peptides are approximately 500 for FAIMS/IMS separations and approximately 10(6) for 3-D FAIMS/IMS/MS, providing a potential platform for ultrahigh-throughput analyses of complex mixtures.     

Alkylating tryptic peptides to enhance electrospray ionization mass spectrometry analysis

A major limitation of mass spectrometry-based proteomics is inefficient and differential ionization during electrospray ionization (ESI). This leads to problems such as increased limits of detection and incomplete sequence coverage of proteins. Incomplete sequence coverage is especially problematic for analyses that require the detection and identification of specific peptides from a protein, such as the analysis of post-translational modifications. We describe here the development and use of aldehyde-based chemistry for the alkylation of peptide primary amines to increase peptide hydrophobicity, providing increased ionization efficiency and concomitant signal enhancement. When employed to modify the peptide products of protein tryptic digests, increased sequence coverage is obtained from combined modified and unmodified digests. To evaluate the utility of alkylation of peptides for selected reaction monitoring (SRM) assays, we alkylated a peptide from the protein Oct4, known to play a role in regulating stem cell differentiation. Increased chromatographic retention and ionization efficiency is observed for the alkylated Oct4 peptide compared to its unmodified form.     

Differentiation of hydroxyproline isomers and isobars in peptides by tandem mass spectrometry

The isomeric 3- and 4-hydroxyprolines are isobaric with the isomers leucine and isoleucine, and all four have, therefore, the same "residue mass" of 113. Secondary fragmentation processes were found that differentiate the hydroxyproline isomers from each other and from the leucines. Variants of synthetic bradykinin containing one or two hydroxyproline moieties were prepared by using manual Edman degradation and/or enzymatic methods. The tandem mass spectra of these peptides were recorded. The C-terminal wn fragment ions allow the differentiation of 4-hydroxyproline from the 3-isomer and isoleucine, while the N-terminal an ions containing 4-hydroxyproline undergo H2O elimination to differentiate this amino acid from the 3-isomer and leucine. Lys-C digestion of a mussel adhesive protein produced a set of decapeptides varying in the degree of hydroxylation of proline and tyrosine. Heterogeneity with respect to 3-hydroxyproline and 4-hydroxyproline at a certain position in these peptides was assessed by tandem mass spectrometry based on the wn ion series in the CID spectra of these Lys-C peptides. Some N-terminal ions further allow for the differentiation of these two isomeric species.     

Profiling signaling peptides in single mammalian cells using mass spectrometry

The peptide content of individual mammalian cells is profiled using matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry. Both enzymatic and nonenzymatic procedures, including a glycerol cell stabilization method, are reported for the isolation of individual mammalian cells in a manner compatible with MALDI MS measurements. Guided microdeposition of MALDI matrix allows samples to be created with suitable analyte-to-matrix ratios. More than 15 peptides are observed in individual rat intermediate pituitary cells. The combination of accurate mass data, expected cleavages by proteolytic enzymes, and postsource decay sequencing allows identification of 14 of these peptides as pro-opiomelanocortin prohormone-derived molecules. These protocols permit the classification of individual mammalian cells by peptide profile, the elucidation of cell-specific prohormone processing, and the discovery of new signaling peptides on a cell-to-cell basis in a wide variety of mammalian cell types.     

Controlling charge states of peptides through inductive electrospray ionization mass spectrometry

A novel ionization device for controlling the charge states of peptides based on an inductive elecrospray ionization technique was developed. This ion source keeps the major capabilities of electrospray ionization (ESI) which is compatible with liquid separation techniques (such as liquid chromatography (LC) and capillary electrophoresis (CE)) and can be potentially used to control the charge states of peptides accurately by simply varying the AC voltage applied. In comparison with conventional ESI, inductive ESI successfully simplifies the mass spectrum by reducing the charge states of peptide to a singly charged one, as well as eliminating the adduct ions.     

High-sensitivity electron capture dissociation tandem FTICR mass spectrometry of microelectrosprayed peptides

Electron capture dissociation (ECD) has previously been shown by other research groups to result in greater peptide sequence coverage than other ion dissociation techniques and to localize labile posttranslational modifications. Here, ECD has been achieved for 10-13-mer peptides microelectrosprayed from 10 nM (10 fmol/microL) solutions and for tryptic peptides from a 50 nM unfractionated digest of a 28-kDa protein. Tandem Fourier transform ion cyclotron resonance (FTICR) mass spectra contain fragment ions corresponding to cleavages at all possible peptide backbone amine bonds, except on the N-terminal side of proline, for substance P and neurotensin. For luteinizing hormone-releasing hormone, all but two expected backbone amine bond cleavages are observed. The tandem FTICR mass spectra of the tryptic peptides contain fragment ions corresponding to cleavages at 6 of 12 (1545.7-Da peptide) and 8 of 21 (2944.5-Da peptide) expected backbone amine bonds. The present sensitivity is 200-2000 times higher than previously reported. These results show promise for ECD as a tool to produce sequence tags for identification of peptides in complex mixtures available only in limited amounts, as in proteomics.     

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.     

Distinguishing and quantifying peptides and proteins containing D-amino acids by tandem mass spectrometry

Tandem mass spectrometry (MS/MS) utilizing both electron capture dissociation (ECD) and collisionally activated dissociation (CAD) was used to develop a qualitative and quantitative analytical method for chiral analysis of individual amino acid residues in polypeptides. ECD produced a more distinct chiral recognition than CAD, which is attributed to the smaller degree of vibrational excitation in ECD. Several peptide and protein model systems were used in this study, including the smallest known protein, tryptophan cage, a lactoferrin peptide, and the biologically relevant opioid peptide, dermorphin. An adaptation of the kinetic method was used to quantify the degree of separation between fragmentation patterns of stereoisomeric peptides as a function of fragment ion abundances. The obtained calibration scale for relative abundances of d-amino acids in diastereomeric peptide mixtures was accurate to 1% for ECD and to 3-5% for CAD. It was found that separation and quantification of stereoisomers could be advantageously performed by nanoflow reversed-phase liquid chromatography, with the objective of on-line MS/MS limited to stereoisomer identification. This technique shows promise for the analysis of chiral substitution in peptides and proteins, broadening the application area for tandem mass spectrometry.