Neuropeptides are essential cell-to-cell signaling messengers and serve important regulatory roles in animals. Although remarkable progress has been made in peptide identification across the Metazoa, for some phyla such as Echinodermata, limited neuropeptides are known and even fewer have been verified on the protein level. We employed peptidomic approaches using bioinformatics and mass spectrometry (MS) to experimentally confirm 23 prohormones and to characterize a new prohormone in nervous system tissue from Strongylocentrotus purpuratus, the purple sea urchin. Ninety-three distinct peptides from known and novel prohormones were detected with MS from extracts of the radial nerves, many of which are reported or experimentally confirmed here for the first time, representing a large-scale study of neuropeptides from the phylum Echinodermata. Many of the identified peptides and their precursor proteins have low homology to known prohormones from other species/phyla and are unique to the sea urchin. By pairing bioinformatics with MS, the capacity to characterize novel peptides and annotate prohormone genes is enhanced.
Neuropeptides are vital for cell-cell communication and function in the regulation of the nervous and endocrine systems. They are generated by post-translational modification (PTM) steps resulting in small active peptides generated from prohormone precursors. Phosphorylation is a significant PTM for the bioactivity of neuropeptides. From the known diversity of distinct neuropeptide functions, it is hypothesized that the extent of phosphorylation varies among different neuropeptides. To assess this hypothesis, neuropeptide-containing dense core secretory vesicles from bovine adrenal medullary chromaffin cells were subjected to global phosphopeptidomics analyses by liquid chromatography (LC)-mass spectrometry (MS/MS). Phosphopeptides were identified directly by LC-MS/MS and indirectly by phosphatase treatment followed by LC-MS/MS. The data identified numerous phosphorylated peptides derived from neuropeptide precursors such as chromogranins, secretogranins, proenkephalin and pro-NPY. Phosphosite occupancies were observed at high and low levels among identified peptides and many of the high occupancy phosphopeptides represent prohormone-derived peptides with currently unknown bioactivities. Peptide sequence analyses demonstrated SxE as the most prevalent phosphorylation site motif, corresponding to phosphorylation sites of the Fam20C protein kinase known to be present in the secretory pathway. The range of high to low phosphosite occupancies for neuropeptides demonstrates cellular regulation of neuropeptide phosphorylation.
A method to facilitate the characterization of stapled or cyclic peptides is reported via an arginine-selective derivatization strategy coupled with MS/MS analysis. Arginine residues are converted to ornithine residues through a deguanidination reaction that installs a highly selectively cleavable site in peptides. Upon activation by CID or UVPD, the ornithine residue cyclizes to promote cleavage of the adjacent amide bond. This Arg-specific process offers a unique strategy for site-selective ring opening of stapled and cyclic peptides. Upon activation of each derivatized peptide, site-specific backbone cleavage at the ornithine residue results in two complementary products: the lactam ring-containing portion of the peptide and the amine-containing portion. The deguanidination process not only provides a specific marker site that initiates fragmentation of the peptide but also offers a means to unlock the staple and differentiate isobaric stapled peptides.
Hydrogen deuterium exchange measured by mass spectrometry (HDX-MS) is a commonly used technique for studying the structural dynamics of proteins in solution. The first part of any bottom-up HDX-MS experiment is to identify the peptides generated from a digestion step. This requires manual inspection of the identified peptides to determine their use for HDX-MS analysis, which is a time-consuming task. Throughout the literature, there have been different approaches for removing peptides that do not yield quantifiable HDX information. This includes using validity scores from the software used in the generation of the peptide map and that the peptide should be found in two out of three technical replicate experiments. Here, we analyze the previously available methods for filtering the identified peptides in regard to their ability to predict whether a peptide will provide quantifiable HDX-MS data or not. We also present a new score-based system relying on a combination of MS/MS parameters that offers an improved method for separating quantifiable peptides from the nonquantifiable. Using this score-based method reduces the number of peptide spectra that needs to be manually inspected and thereby the time spent curating HDX-MS data.
High-accuracy MS/MS spectra of deprotonated ions of 390 dipeptides and 137 peptides with three to six residues are studied. Many amino acid residues undergo neutral losses from their side chains. The most abundant is the loss of acetaldehyde from threonine. The abundance of losses from the side chains of other amino acids is estimated relative to that of threonine. While some amino acids lose the whole side chain, others lose only part of it, and some exhibit two or more different losses. Side-chain neutral losses are less abundant in the spectra of protonated peptides, being significant mainly for methionine and arginine. In addition to the neutral losses, many amino acid residues in deprotonated peptides produce specific negative ions after peptide bond cleavage. An expanded list of fragment ions from protonated peptides is also presented and compared with those of deprotonated peptides. Fragment ions are mostly different for these two cases. These lists of fragments are used to annotate peptide mass spectral libraries and to aid in the confirmation of specific amino acids in peptides.
Immonium ions are commonly observed in the high energy fragmentation of peptide ions. In a MALDI-TOF/TOF mass spectrometer, singly charged peptides photofragmented with 157 nm VUV light yield a copious abundance of immonium ions, especially those from aromatic residues. However, their intensities may vary from one peptide to another. In this work, the effect of varying amino acid position, peptide length, and peptide composition on immonium ion yield is investigated. Internal immonium ions are found to have the strongest intensity, whereas immonium ions arising from C-terminal residues are the weakest. Peptide length and competition among residues also strongly influence the immonium ion production. Quantum calculations provide insights about immonium ion structures and the fragment ion conformations that promote or inhibit immonium ion formation.
The D-residues are crucial to biological function of D-amino acid containing peptides (DAACPs). Previous ion mobility mass spectrometry (IM-MS) studies revealing oligomerization patterns of amyloid cascade demonstrated conversion from native soluble unstructured assembly to fibril ß-sheet oligomers, which has been implicated in amyloid diseases, such as Alzheimer’s disease and type 2 diabetes. Although neuropeptides are typically present at very low concentrations in circulation, their local concentrations could be much higher in large dense core vesicles, forming dimers or oligomers. We studied the oligomerization of protonated and metal-adducted achatin I and dermorphin peptide isomers with IM-MS. Our results suggested that dimerization, oligomerization, and metal adduction augment the structural differences between D/L peptide isomers compared to protonated monomers. Dimers and oligomers enhanced the structural differences between D/L peptide isomers in both aqueous and organic solvent system. Furthermore, some oligomer forms were only observed for either D- or L-isomers, indicating the importance of chiral center in oligomerization process. The oligomerization patterns of D/L isomers appear to be similar. Potassium adducts were detected to enlarge the structural differences between D/L isomers.
Neuropeptides are important signaling molecules used by nervous systems to mediate and fine-tune neuronal communication. They can function as neurotransmitters or neuromodulators in neural circuits, or they can be released as neurohormones to target distant cells and tissues. Neuropeptides are typically cleaved from larger precursor proteins by the action of proteases and can be the subject of post-translational modifications. The short, mature neuropeptide sequences often entail the only evolutionarily reasonably conserved regions in these precursor proteins. Therefore, it is particularly challenging to predict all putative bioactive peptides through in silico mining of neuropeptide precursor sequences. Peptidomics is an approach that allows de novo characterization of peptides extracted from body fluids, cells, tissues, organs, or whole-body preparations. Mass spectrometry, often combined with on-line liquid chromatography, is a hallmark technique used in peptidomics research. Here, we used an acidified methanol extraction procedure and a quadrupole-Orbitrap LC-MS/MS pipeline to analyze the neuropeptidome of Caenorhabditis elegans. We identified an unprecedented number of 203 mature neuropeptides from C. elegans whole-body extracts, including 35 peptides from known, hypothetical, as well as from completely novel neuropeptide precursor proteins that have not been predicted in silico. This set of biochemically verified peptide sequences provides the most elaborate C. elegans reference neurpeptidome so far. To exploit this resource to the fullest, we make our in-house database of known and predicted neuropeptides available to the community as a valuable resource. We are providing these collective data to help the community progress, amongst others, by supporting future differential and/or functional studies.
Calibrants based on synthetic dendrimers have been recently proposed as a versatile alternative to peptides and proteins for both MALDI and ESI mass spectrometry calibration. Because of their modular synthetic platform, dendrimer calibrants are particularly amenable to tailoring for specific applications. Utilizing this versatility, a set of dendrimers has been designed as an internal calibrant with a tailored mass defect to differentiate them from the majority of natural peptide analytes. This was achieved by incorporating a tris-iodinated aromatic core as an initiator for the dendrimer synthesis, thereby affording multiple calibration points (m/z range 600–2300) with an optimized mass-defect offset relative to all peptides composed of the 20 most common proteinogenic amino acids.
The present study demonstrates that one-step peptide backbone fragmentations can be achieved using the TEMPO [2-(2,2,6,6-tetramethyl piperidine-1-oxyl)]-assisted free radical-initiated peptide sequencing (FRIPS) mass spectrometry in a hybrid quadrupole time-of-flight (Q-TOF) mass spectrometer and a Q-Exactive Orbitrap instrument in positive ion mode, in contrast to two-step peptide fragmentation in an ion-trap mass spectrometer (reference Anal. Chem. 85, 7044–7051 (30)). In the hybrid Q-TOF and Q-Exactive instruments, higher collisional energies can be applied to the target peptides, compared with the low collisional energies applied by the ion-trap instrument. The higher energy deposition and the additional multiple collisions in the collision cell in both instruments appear to result in one-step peptide backbone dissociations in positive ion mode. This new finding clearly demonstrates that the TEMPO-assisted FRIPS approach is a very useful tool in peptide mass spectrometry research.
The rising profile of ion mobility spectrometry (IMS) in proteomics has driven the efforts to predict peptide cross-sections. In the simplest approach, these are derived by adding the contributions of all amino acid residues and post-translational modifications (PTMs) defined by their intrinsic size parameters (ISPs). We show that the ISPs for PTMs can be calculated from properties of constituent atoms, and introduce the “impact scores” that govern the shift of cross-sections from the central mass-dependent trend for unmodified peptides. The ISPs and scores tabulated for 100 more common PTMs enable predicting the domains for modified peptides in the IMS/MS space that would guide subproteome investigations.
Peptides with deamidated asparagine residues and oxidized methionine residues are often not resolved sufficiently to allow quantitation of their native and modified forms using reversed phase (RP) chromatography. The accurate quantitation of these modifications is vital in protein biotherapeutic analysis because they can affect a protein’s function, activity, and stability. We demonstrate here that hydrophilic interaction liquid chromatography (HILIC) adequately and predictably separates peptides with these modifications from their native counterparts. Furthermore, coefficients describing the extent of the hydrophilicity of these modifications have been derived and were incorporated into a previously made peptide retention prediction model that is capable of predicting the retention times of peptides with and without these modifications.
Food intake is regulated by various neuromodulators, including numerous neuropeptides. However, it remains elusive at the molecular and cellular level as to how these important chemicals regulate internal processes and which regions of the neuronal organs are responsible for regulating the behavior. Here we report a comparative neuropeptidomic analysis of the brain and pericardial organ (PO) in response to feeding in two well-studied crustacean physiology model organisms, Callinectes sapidus and Carcinus maenas, using mass spectrometry (MS) techniques. A multifaceted MS-based approach has been developed to obtain complementary information on the expression changes of a large array of neuropeptides in the brain and PO. The method employs stable isotope labeling of brain and PO extracts for relative MS quantitation, capillary electrophoresis (CE)-MS for fractionation and high-specificity analysis, and mass spectrometric imaging (MSI) for in-situ molecular mapping of peptides. A number of neuropeptides, including RFamides, B-type allatostatins (AST-B), RYamides, and orcokinins exhibit significant changes in abundance after feeding in this investigation. Peptides from the AST-B family found in PO tissue were shown to have both altered expression and localization changes after feeding, indicating that they may be a class of vital neuropeptide regulators involved in feeding behavior.
A high resolving power shotgun lipidomics strategy using gas-phase fractionation and data-dependent acquisition (DDA) was applied toward comprehensive characterization of lipids in a hen ovarian tissue in an untargeted fashion. Using this approach, a total of 822 unique lipids across a diverse range of lipid categories and classes were identified based on their MS/MS fragmentation patterns. Classes of glycerophospholipids and glycerolipids, such as glycerophosphocholines (PC), glycerophosphoethanolamines (PE), and triglycerides (TG), are often the most abundant peaks observed in shotgun lipidomics analyses. These ions suppress the signal from low abundance ions and hinder the chances of characterizing low abundant lipids when DDA is used. These issues were circumvented by utilizing gas-phase fractionation, where DDA was performed on narrow m/z ranges instead of a broad m/z range. Employing gas-phase fractionation resulted in an increase in sensitivity by more than an order of magnitude in both positive- and negative-ion modes. Furthermore, the enhanced sensitivity increased the number of lipids identified by a factor of ≈4, and facilitated identification of low abundant lipids from classes such as cardiolipins that are often difficult to observe in untargeted shotgun analyses and require sample-specific preparation steps prior to analysis. This method serves as a resource for comprehensive profiling of lipids from many different categories and classes in an untargeted manner, as well as for targeted and quantitative analyses of individual lipids. Furthermore, this comprehensive analysis of the lipidome can serve as a species- and tissue-specific database for confident identification of other MS-based datasets, such as mass spectrometry imaging.
Matrix-assisted laser desorption/ionization (MALDI) in-source decay was studied in the negative ion mode on deprotonated peptides to determine its usefulness for obtaining extensive sequence information for acidic peptides. Eight biological acidic peptides, ranging in size from 11 to 33 residues, were studied by negative ion mode ISD (nISD). The matrices 2,5-dihydroxybenzoic acid, 2-aminobenzoic acid, 2-aminobenzamide, 1,5-diaminonaphthalene, 5-amino-1-naphthol, 3-aminoquinoline, and 9-aminoacridine were used with each peptide. Optimal fragmentation was produced with 1,5-diaminonphthalene (DAN), and extensive sequence informative fragmentation was observed for every peptide except hirudin(54–65). Cleavage at the N–Cα bond of the peptide backbone, producing c′ and z′ ions, was dominant for all peptides. Cleavage of the N–Cα bond N-terminal to proline residues was not observed. The formation of c and z ions is also found in electron transfer dissociation (ETD), electron capture dissociation (ECD), and positive ion mode ISD, which are considered to be radical-driven techniques. Oxidized insulin chain A, which has four highly acidic oxidized cysteine residues, had less extensive fragmentation. This peptide also exhibited the only charged localized fragmentation, with more pronounced product ion formation adjacent to the highly acidic residues. In addition, spectra were obtained by positive ion mode ISD for each protonated peptide; more sequence informative fragmentation was observed via nISD for all peptides. Three of the peptides studied had no product ion formation in ISD, but extensive sequence informative fragmentation was found in their nISD spectra. The results of this study indicate that nISD can be used to readily obtain sequence information for acidic peptides.
Disulfide bond formation is critical for maintaining structure stability and function of many peptides and proteins. Mass spectrometry has become an important tool for the elucidation of molecular connectivity. However, the interpretation of the tandem mass spectral data of disulfide-linked peptides has been a major challenge due to the lack of appropriate tools. Developing proper data analysis software is essential to quickly characterize disulfide-linked peptides. A thorough and in-depth understanding of how disulfide-linked peptides fragment in mass spectrometer is a key in developing software to interpret the tandem mass spectra of these peptides. Two model peptides with inter- and intra-chain disulfide linkages were used to study fragmentation behavior in both collisional-activated dissociation (CAD) and electron-based dissociation (ExD) experiments. Fragments generated from CAD and ExD can be categorized into three major types, which result from different S–S and C–S bond cleavage patterns. DiSulFinder is a computer algorithm that was newly developed based on the fragmentation observed in these peptides. The software is vendor neutral and capable of quickly and accurately identifying a variety of fragments generated from disulfide-linked peptides. DiSulFinder identifies peptide backbone fragments with S–S and C–S bond cleavages and, more importantly, can also identify fragments with the S–S bond still intact to aid disulfide linkage determination. With the assistance of this software, more comprehensive disulfide connectivity characterization can be achieved.
Our scientific interests involve de novo sequencing of non-tryptic natural amphibian skin peptides including those with intramolecular S–S bond by means of exclusively mass spectrometry. Reliable discrimination of the isomeric leucine/isoleucine residues during peptide sequencing by means of mass spectrometry represents a bottleneck in the workflow for complete automation of the primary structure elucidation of these compounds. MS3 is capable of solving the problem. Earlier we demonstrated the advanced efficiency of ETD-HCD method to discriminate Leu/Ile in individual peptides by consecutive application of ETD to the polyprotonated peptides followed by HCD applied to the manually selected primary z-ions with the targeted isomeric residues at their N-termini and registration of the characteristic w-ions. Later this approach was extended to deal with several (4–7) broad band mass ranges, without special isolation of the primary z-ions. The present paper demonstrates an advanced version of this method when EThcD is applied in the whole mass range to a complex mixture of natural non-tryptic peptides without their separation and intermediate isolation of the targeted z-ions. The proposed EThcD method showed over 81% efficiency for the large natural peptides with intact disulfide ring, while the interfering process of radical site migration is suppressed. Due to higher speed and sensitivity, the proposed EThcD approach facilitates the analytical procedure and allows for the automation of the entire experiment and data processing. Moreover, in some cases it gives a chance to establish the nature of the residues in the intact intramolecular disulfide loops.
The small ubiquitin-like modifier (SUMO) is a member of the family of ubiquitin-like modifiers (UBLs) and is involved in important cellular processes, including DNA damage response, meiosis and cellular trafficking. The large-scale identification of SUMO peptides in a site-specific manner is challenging not only because of the low abundance and dynamic nature of this modification, but also due to the branched structure of the corresponding peptides that further complicate their identification using conventional search engines. Here, we exploited the unusual structure of SUMO peptides to facilitate their separation by high-field asymmetric waveform ion mobility spectrometry (FAIMS) and increase the coverage of SUMO proteome analysis. Upon trypsin digestion, branched peptides contain a SUMO remnant side chain and predominantly form triply protonated ions that facilitate their gas-phase separation using FAIMS. We evaluated the mobility characteristics of synthetic SUMO peptides and further demonstrated the application of FAIMS to profile the changes in protein SUMOylation of HEK293 cells following heat shock, a condition known to affect this modification. FAIMS typically provided a 10-fold improvement of detection limit of SUMO peptides, and enabled a 36% increase in SUMO proteome coverage compared to the same LC-MS/MS analyses performed without FAIMS.
Characterization of acidic peptides and proteins is greatly hindered due to lack of suitable analytical techniques. Here we present the implementation of 213 nm ultraviolet photodissociation (UVPD) in high-resolution quadrupole-Orbitrap mass spectrometer in negative polarity for peptide anions. Radical-driven backbone fragmentation provides 22 distinctive fragment ion types, achieving the complete sequence coverage for all reported peptides. Hydrogen-deficient radical anion not only promotes the cleavage of Cα–C bond but also stimulates the breaking of N–Cα and C–N bonds. Radical-directed loss of small molecules and specific side chain of amino acids are detected in these experiments. Radical containing side chain of amino acids (Tyr, Ser, Thr, and Asp) may possibly support the N–Cα backbone fragmentation. Proline comprising peptides exhibit the unusual fragment ions similar to reported earlier. Interestingly, basic amino acids such as Arg and Lys also stimulated the formation of abundant b and y ions of the related peptide anions. Loss of hydrogen atom from the charge-reduced radical anion and fragment ions are rationalized by time-dependent density functional theory (TDDFT) calculation, locating the potential energy surface (PES) of ππ* and repulsive πσ* excited states of a model amide system.
Protein footprinting combined with mass spectrometry provides a method to study protein structures and interactions. To improve further current protein footprinting methods, we adapted the fast photochemical oxidation of proteins (FPOP) platform to utilize carbenes as the footprinting reagent. A Nd-YAG laser provides 355 nm laser for carbene generation in situ from photoleucine as the carbene precursor in a flow system with calmodulin as the test protein. Reversed-phase liquid chromatography coupled with mass spectrometry is appropriate to analyze the modifications produced in this footprinting. By comparing the modification extent of apo and holo calmodulin on the peptide level, we can resolve different structural domains of the protein. Carbene footprinting in a flow system is promising.
