Unexpected Crosslinking and Diglycation as Advanced Glycation End-Products from Glyoxal

Glyoxal-derived advanced glycation end-products (AGEs) are formed in physiological systems affecting protein/peptide function and structure. These AGEs are generated during aging and chronic diseases such as diabetes and are considered arginine glycating agents. Thus, the study of glyoxal-derived AGEs in lysine residues and amino acid competition is addressed here using acetylated and non-acetylated undecapeptides, with one arginine and one lysine residue available for glycation. Tandem mass spectrometry results from a Fourier transform ion cyclotron resonance mass spectrometer showed glycated species at both the arginine and lysine residues. One species with the mass addition of 116.01096 Da is formed at the arginine residue. A possible structure is proposed to explain this finding (Nδ-[2-(dihydroxymethyl)-2H,3aH,4H,6aH-[1, 3]dioxolo[5,6-d]imidazolin-5-yl]-L-ornithine-derived AGE). The second species corresponded to intramolecular crosslink involving the lysine residue and its presence is checked with ion-mobility mass spectrometry. Graphical Abstract

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Selective Detection of Carbohydrates and Their Peptide Conjugates by ESI-MS Using Synthetic Quaternary Ammonium Salt Derivatives of Phenylboronic Acids

We present new tags based on the derivatives of phenylboronic acid and apply them for the selective detection of sugars and peptide-sugar conjugates in mass spectrometry. We investigated the binding of phenylboronic acid and its quaternary ammonium salt (QAS) derivatives to carbohydrates and peptide-derived Amadori products by HR-MS and MS/MS experiments. The formation of complexes between sugar or sugar-peptide conjugates and synthetic tags was confirmed on the basis of the unique isotopic distribution resulting from the presence of boron atom. Moreover, incorporation of a quaternary ammonium salt dramatically improved the efficiency of ionization in mass spectrometry. It was found that the formation of a complex with phenylboronic acid stabilizes the sugar moiety in glycated peptides, resulting in simplification of the fragmentation pattern of peptide-derived Amadori products. The obtained results suggest that derivatization of phenylboronic acid as QAS is a promising method for sensitive ESI-MS detection of carbohydrates and their conjugates formed by non-enzymatic glycation or glycosylation.

Study of an Unusual Advanced Glycation End-Product (AGE) Derived from Glyoxal Using Mass Spectrometry

Glycation is a post-translational modification (PTM) that affects the physiological properties of peptides and proteins. In particular, during hyperglycaemia, glycation by α-dicarbonyl compounds generate α-dicarbonyl-derived glycation products also called α-dicarbonyl-derived advanced glycation end products. Glycation by the α-dicarbonyl compound known as glyoxal was studied in model peptides by MS/MS using a Fourier transform ion cyclotron resonance mass spectrometer. An unusual type of glyoxal-derived AGE with a mass addition of 21.98436 Da is reported in peptides containing combinations of two arginine-two lysine, and one arginine-three lysine amino acid residues. Electron capture dissociation and collisionally activated dissociation results supported that the unusual glyoxal-derived AGE is formed at the guanidino group of arginine, and a possible structure is proposed to illustrate the 21.9843 Da mass addition.

ECD of Tyrosine Phosphorylation in a Triple Quadrupole Mass Spectrometer with a Radio-Frequency-Free Electromagnetostatic Cell

A radio frequency-free electromagnetostatic (EMS) cell devised for electron-capture dissociation (ECD) of ions has been retrofitted into the collision-induced dissociation (CID) section of a triple quadrupole mass spectrometer to enable recording of ECD product-ion mass spectra and simultaneous recording of ECD-CID product-ion mass spectra. This modified instrument can be used to produce easily interpretable ECD and ECD-CID product-ion mass spectra of tyrosine-phosphorylated peptides that cover over 50% of their respective amino-acid sequences and readily identify their respective sites of phosphorylation. ECD fragmentation of doubly protonated, tyrosine-phosphorylated peptides, which was difficult to observe with FT-ICR instruments, occurs efficiently in the EMS cell. Figure

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In-Depth Comparative Characterization of Hemoglobin Glycation in Normal and Diabetic Bloods by LC-MSMS

The glycation level at β-Val-1 of the hemoglobin β chain in human blood (HbA1c%) is used to diagnose diabetes and other diseases. However, hemoglobin glycation occurs on multiple sites on different isoforms with different kinetics, but its differential profile has not been clearly demonstrated. In this study, hemoglobin was extracted from the blood of normal and diabetic individuals by protein precipitation. Triplicate solutions prepared from each sample were directly analyzed or digested with multiple enzymes and then analyzed by nano-LC/MS via bottom-up approach for side-by-side characterization. Intact hemoglobin analysis indicated a single glucose-dominant glycation, which showed good correlation with the HbA1c% values. Moreover, full sequence (100 %) of α/β globin was mapped and seven glycation sites were unambiguously assigned. In addition to β-Val-1, two other major sites at α-Lys-61 and β-Lys-66, which contain the common sequence HGKK, and four minor sites (<1 %) on α-Val-1, β-Lys-132, α-Lys-127, and α-Lys-40 were identified. All sites were shown to exhibit similar patterns of site distribution despite different glucose levels. Both the intact mass measurement and bottom-up data consistently indicated that the total glycation percentage of the β-globin was twice higher than the α-globin. Using molecular modeling, the 3D structure of the consensus sequence (HGKK) was shown to contain a phosphate triangle cavity, which helps to catalyze the glycation reaction. For the first time, hemoglobin glycation in normal and diabetic bloods was comparatively characterized in-depth with 100 % sequence coverage. The results provide insight about the HbA1c parameter and help define the new and old markers.

Improved Identification and Relative Quantification of Sites of Peptide and Protein Oxidation for Hydroxyl Radical Footprinting

Protein oxidation is typically associated with oxidative stress and aging and affects protein function in normal and pathological processes. Additionally, deliberate oxidative labeling is used to probe protein structure and protein–ligand interactions in hydroxyl radical protein footprinting (HRPF). Oxidation often occurs at multiple sites, leading to mixtures of oxidation isomers that differ only by the site of modification. We utilized sets of synthetic, isomeric “oxidized” peptides to test and compare the ability of electron-transfer dissociation (ETD) and collision-induced dissociation (CID), as well as nano-ultra high performance liquid chromatography (nanoUPLC) separation, to quantitate oxidation isomers with one oxidation at multiple adjacent sites in mixtures of peptides. Tandem mass spectrometry by ETD generates fragment ion ratios that accurately report on relative oxidative modification extent on specific sites, regardless of the charge state of the precursor ion. Conversely, CID was found to generate quantitative MS/MS product ions only at the higher precursor charge state. Oxidized isomers having multiple sites of oxidation in each of two peptide sequences in HRPF product of protein Robo-1 Ig1-2, a protein involved in nervous system axon guidance, were also identified and the oxidation extent at each residue was quantified by ETD without prior liquid chromatography (LC) separation. ETD has proven to be a reliable technique for simultaneous identification and relative quantification of a variety of functionally different oxidation isomers, and is a valuable tool for the study of oxidative stress, as well as for improving spatial resolution for HRPF studies.

Label Scrambling During CID of Covalently Labeled Peptide Ions

Covalent labeling along with mass spectrometry is finding more use as a means of studying the higher order structure of proteins and protein complexes. Diethylpyrocarbonate (DEPC) is an increasingly used reagent for these labeling experiments because it is capable of modifying multiple residues at the same time. Pinpointing DEPC-labeled sites on proteins is typically needed to obtain more resolved structural information, and tandem mass spectrometry after protein proteolysis is often used for this purpose. In this work, we demonstrate that in certain instances, scrambling of the DEPC label from one residue to another can occur during collision-induced dissociation (CID) of labeled peptide ions, resulting in ambiguity in label site identity. From a preliminary study of over 30 labeled peptides, we find that scrambling occurs in about 25% of the peptides and most commonly occurs when histidine residues are labeled. Moreover, this scrambling appears to occur more readily under non-mobile proton conditions, meaning that low charge-state peptide ions are more prone to this reaction. For all peptides, we find that scrambling does not occur during electron transfer dissociation, which suggests that this dissociation technique is a safe alternative to CID for correct label site identification. Graphical Abstract

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Identification and relative quantification of specific glycation sites in human serum albumin

Glycation (or non-enzymatic glycosylation) is a common non-enzymatic covalent modification of human proteins. Glucose, the highest concentrated monosaccharide in blood, can reversibly react with amino groups of proteins to form Schiff bases that can rearrange to form relatively stable Amadori products. These can be further oxidized to advanced glycation end products (AGEs). Here, we analyzed the glycation patterns of human serum albumin (HSA) in plasma samples obtained from five patients with type 2 diabetes mellitus. Therefore, glycated peptides from a tryptic digest of plasma were enriched with m-aminophenylboronic acid (mAPBA) affinity chromatography. The glycated peptides were then further separated in the second dimension by RP-HPLC coupled on-line to an electrospray ionization (ESI) tandem mass spectrometer (MS/MS). Altogether, 18 Amadori peptides, encompassing 40% of the HSA sequence, were identified. The majority of the peptides were detected and relatively quantified in all five samples with a high reproducibility among the replicas. Eleven Lys-residues were glycated at similar quantities in all samples, with glycation site Lys₅₄₉ (K_Am(Glc)QTALVELVK) being the most abundant. In conclusion, the established mAPBA/nanoRP-HPLC-ESI-MS/MS approach could reproducibly identify and quantify glycation sites in plasma samples, potentially useful in diagnosis and therapeutic control.     

Peptide Dimethylation: Fragmentation Control via Distancing the Dimethylamino Group

Direct reductive methylation of peptides is a common method for quantitative proteomics. It is an active derivatization technique; with participation of the dimethylamino group, the derivatized peptides preferentially release intense a₁ ions. The advantageous generation of a₁ ions for quantitative proteomic profiling, however, is not desirable for targeted proteomic quantitation using multiple reaction monitoring mass spectrometry; this mass spectrometric method prefers the derivatizing group to stay with the intact peptide ions and multiple fragments as passive mass tags. This work investigated collisional fragmentation of peptides whose amine groups were derivatized with five linear ω-dimethylamino acids, from 2-(dimethylamino)-acetic acid to 6-(dimethylamino)-hexanoic acid. Tandem mass spectra of the derivatized tryptic peptides revealed different preferential breakdown pathways. Together with energy resolved mass spectrometry, it was found that shutting down the active participation of the terminal dimethylamino group in fragmentation of derivatized peptides is possible. However, it took a separation of five methylene groups between the terminal dimethylamino group and the amide formed upon peptide derivatization. For the first time, the gas-phase fragmentation of peptides derivatized with linear ω-dimethylamino acids of systematically increasing alkyl chain lengths is reported. Figure

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Electron Capture Dissociation and Collision Induced Dissociation of S-Dipalmitoylated Peptides

Here we investigate the effect of S-dipalmitoylation on the electron capture dissociation (ECD) behavior of peptides. The ECD and collision induced dissociation (CID) of peptides modified by covalent attachment of [(RS)-2,3-di(palmitoyloxy)-propyl] (PAM2) group to cysteine residues [C(PAM2)LEYDTGFK and RPPGC(PAM2)SPFK] were examined. The results suggest that ECD of S-dipalmitoylated peptides can provide both primary sequence information and structural information regarding the modification. The structural information provided by CID is complementary to that provided by ECD.

Site-specific qualitative and quantitative analysis of the N- and O-glycoforms in recombinant human erythropoietin

Recombinant human erythropoietin (rhEPO) has been extensively used as a pharmaceutical product for treating anemia. Glycosylation of rhEPO affects the biological activity, immunogenicity, pharmacokinetics, and in-vivo clearance rate of rhEPO. Characterization of the glycosylation status of rhEPO is of great importance for quality control. In this study, we established a fast and comprehensive approach for reliable characterization and relative quantitation of rhEPO glycosylation, which combines multiple-enzyme digestion, hydrophilic-interaction chromatography (HILIC) enrichment of glycopeptides, and tandem mass spectrometry (MS) analysis. The N-linked and O-linked intact glycopeptides were analyzed with high-resolution and high-accuracy (HR–AM) mass spectrometry using an Orbitrap. In total, 74 intact glycopeptides from four glycosylation sites at N₂₄, N₃₈, N₈₃, and O₁₂₆ were identified, with the simultaneous determination of peptide sequences and glycoform compositions. The extracted ion chromatograms based on the HR–AM data enabled relative quantification of glycoforms. Our results could be extended to quality control of rhEPO or could help establish detection approaches for glycosylation of other proteins. Graphical Abstract

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Structural stability–chromatographic retention relationship on exenatide diastereomer separation

In this study, the relationship of the structural stability of peptide diastereomers in elution solvents and their retention behaviors in reversed-phase chromatography (RPC) was examined to provide guidance on the solvent selection for a better separation of peptide diastereomers. We investigated the chromatographic retention behaviors of exenatide, a peptide drug for the treatment of type II diabetes mellitus and its three diastereomers using RPC and implicit molecular dynamics (MD) simulation analysis. Three diastereomers involved in the single serine residue mutation of d-form at the 11th, 32nd, and 39th residues were investigated in this study. Results show that the order of the solution structural stability of exenatide and its diastereomers is consistent with their retention order by 36?% acetonitrile/water elution. The sample loading solvent also affects the retention behaviors of exenatide peptide diastereomers in RPC column. Furthermore, a larger solution conformation energy difference of the critical pair of exenatide and its diastereomer (d-Ser39) at the elution solvent of 32?% tetrahydrofuran/water were obtained by MD simulation, and baseline separation was proved experimentally. In summary, we demonstrated that the solution structural stability–chromatographic retention relationship could be a powerful tool for elution solvent selection in peptide chromatographic purification, especially valuable for the separation of critical pair of diastereomers.

Direct Analysis of Large Living Organism by Megavolt Electrostatic Ionization Mass Spectrometry

A new ambient ionization method allowing the direct chemical analysis of living human body by mass spectrometry (MS) was developed. This MS method, namely Megavolt Electrostatic Ionization Mass Spectrometry, is based on electrostatic charging of a living individual to megavolt (MV) potential, illicit drugs, and explosives on skin/glove, flammable solvent on cloth/tissue paper, and volatile food substances in breath were readily ionized and detected by a mass spectrometer. Figure

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Isolation and sequence analysis of peptides from the skin secretion of the Middle East tree frog Hyla savignyi

Novel peptides were identified in the skin secretion of the tree frog Hyla savignyi. Skin secretions were collected by mild electrical stimulation. Peptides were separated by reversed-phase high-performance liquid chromatography. Mass spectra were acquired by electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS), and fragment ion spectra were obtained after collision-induced dissociation and electron capture dissociation. Peptides were analyzed by manual de novo sequencing and composition-based sequencing (CBS). Sequence analyses of three so far undescribed, structurally unrelated peptides are presented in this paper, having the sequences DDSEEEEVE-OH, P*EEVEEERJK-OH, and GJJDPJTGJVGGJJ-NH₂. The glutamate-rich sequences are assumed to be acidic spacer peptides of the prepropeptide. One of these peptides contains the modified amino acid hydroxyproline, as identified and localized by high-accuracy FTICR-MS. Combination of CBS and of experience-based manual sequence analysis as complementary and database-independent sequencing strategies resulted in peptide identification with high reliability.

Quantitative proteomics reveals the kinetics of trypsin-catalyzed protein digestion

Trypsin is the popular protease to digest proteins into peptides in shotgun proteomics, but few studies have attempted to systematically investigate the kinetics of trypsin-catalyzed protein digestion in proteome samples. In this study, we applied quantitative proteomics via triplex stable isotope dimethyl labeling to investigate the kinetics of trypsin-catalyzed cleavage. It was found that trypsin cleaves the C-terminal to lysine (K) and arginine (R) residues with higher rates for R. And the cleavage sites surrounded by neutral residues could be quickly cut, while those with neighboring charged residues (D/E/K/R) or proline residue (P) could be slowly cut. In a proteome sample, a huge number of proteins with different physical chemical properties coexists. If any type of protein could be preferably digested, then limited digestion could be applied to reduce the sample complexity. However, we found that protein abundance and other physicochemical properties, such as molecular weight (Mw), grand average of hydropathicity (GRAVY), aliphatic index, and isoelectric point (pI) have no notable correlation with digestion priority of proteins. Graphical Abstract

Sequence logos of four cleavage site types with different kinetics (very fast, fast, slow, and very slow sites)     

Charge Transfer Dissociation (CTD) Mass Spectrometry of Peptide Cations Using Kiloelectronvolt Helium Cations

A kiloelectronvolt beam of helium ions is used to ionize and fragment precursor peptide ions starting in the 1+ charge state. The electron affinity of helium cations (24.6 eV) exceeds the ionization potential of protonated peptides and can therefore be used to abstract an electron from—or charge exchange with—the isolated precursor ions. Kiloelectronvolt energies are used, (1) to overcome the Coulombic repulsion barrier between the cationic reactants, (2) to overcome ion-defocussing effects in the ion trap, and (3) to provide additional activation energy. Charge transfer dissociation (CTD) of the [M+H]⁺ precursor of Substance P gives product ions such as [M+H]^2+? and a dominant series of a ions in both the 1+ and 2+ charge states. These observations, along with the less-abundant a + 1 ions, are consistent with ultraviolet photodissociation (UVPD) results of others and indicate that C–C_α cleavages are possible through charge exchange with helium ions. Although the efficiencies and timescale of CTD are not yet suitable for on-line chromatography, this new approach to ion activation provides an additional potential tool for the interrogation of gas phase ions. Graphical Abstract

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Automated Deconvolution of Overlapped Ion Mobility Profiles

Presence of unresolved ion mobility (IM) profiles limits the efficient utilization of IM mass spectrometry (IM-MS) systems for isomer differentiation. Here, we introduce an automated ion mobility deconvolution (AIMD) computer software for streamlined deconvolution of overlapped IM-MS profiles. AIMD is based on a previously reported post-IM/collision-induced dissociation (CID) deconvolution approach [J. Am. Soc. Mass Spectrom. 23, 1873 (2012)] and, unlike the previously reported manual approach, it does not require resampling of post-IM/CID data. A novel data preprocessing approach is utilized to improve the accuracy and efficiency of the deconvolution process. Results from AIMD analysis of overlapped IM profiles of data from (1) Waters Synapt G1 for a binary mixture of isomeric peptides (amino acid sequences: GRGDS and SDGRG) and (2) Waters Synapt G2-S for a binary mixture of isomeric trisaccharides (raffinose and isomaltotriose) are presented. Graphical Abstract

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Comparative evaluation of extraction methods for simultaneous mass-spectrometric analysis of complex lipids and primary metabolites from human blood plasma

Metabolomic results on human blood plasma largely depend on the sample preparation protocols employed for protein precipitation and metabolite extraction. Five different extraction methods were examined, which can be grouped into two categories, liquid-liquid extraction and protein precipitation methods, including long-standing protocols such as the Folch extraction and Bligh-Dyer extraction in comparison to modern methods such as the Matyash protocol and two global metabolite extraction methods. Extracts were subjected to analysis of blood plasma lipids and primary metabolites by using chip-based direct infusion nanoelectrospray tandem mass spectrometry and gas chromatography coupled to time-of-flight mass spectrometry, respectively. Optimal extraction schemes were evaluated based on the number of identified metabolites, extraction efficiency, compound diversity, reproducibility, and convenience for high-throughput sample preparations. Results showed that Folch and Matyash methods were equally valid and robust for lipidomic assessments while primary metabolites were better assessed by the protein precipitation methods with organic solvent mixtures. Graphical Abstract

Schematic workflow of five extraction methods and subsequent mass spectrometry analysis using GC-TOF MS and nanoelectrospray direct-infusion ion trap MS/MS?