Nonenzymatic Posttranslational Modifications and Peptide Cleavages Observed in Peptide Epimers.

Posttranslational modifications (PTMs) play vital roles in cellular homeostasis and are implicated in various pathological conditions. This work uses two ion mobility spectrometry-mass spectrometry (IMS-MS) modalities, drift-tube IMS (DT-IMS) and trapped IMS (TIMS), to characterize three important nonenzymatic PTMs that induce no mass loss: l/d isomerization, aspartate/isoaspartate isomerization, and cis/trans proline isomerization. These PTMs are assessed in a single peptide system, the recently discovered pleurin peptides, Plrn2, from Aplysia californica. We determine that the DT-IMS-MS/MS can capture and locate asparagine deamidation into aspartate and its subsequent isomerization to isoaspartate, a key biomarker for age-related diseases. Additionally, nonenzymatic peptide cleavage via in-source fragmentation is evaluated for differences in the intensities and patterns of fragment peaks between these PTMs. Peptide fragments resulting from in-source fragmentation, preceded by peptide denaturation by liquid chromatography (LC) mobile phase, exhibited cis/trans proline isomerization. Finally, the effects of differing the fragmentation voltage at the source and solution-based denaturation conditions on in-source fragmentation profiles are evaluated, confirming that LC denaturation and in-source fragmentation profoundly impact N-terminal peptide bond cleavages of Plrn2 and the structures of their fragment ions. With that, LC-IMS-MS/MS coupled with in-source fragmentation could be a robust method to identify three important posttranslational modifications: l/d isomerization, Asn-deamidation leading to Asp/IsoAsp isomerization, and cis/trans proline isomerization.

Analysis of Peptide Stereochemistry in Single Cells by Capillary Electrophoresis-Trapped Ion Mobility Spectrometry Mass Spectrometry.

Single cell analysis strives to probe molecular heterogeneity in morphologically similar cell populations through quantitative or qualitative measurements of genetic, proteomic, or metabolic products. Here, we applied mass analysis of single neurons to investigate cell-cell signaling peptides. The multiplicity of endogenous cell-cell signaling peptides is a common source of chemical diversity among cell populations. Certain peptides can undergo post-translational isomerization of select residues, which has important physiological consequences. The limited number of single cell analysis techniques that are sensitive to peptide stereochemistry make it challenging to study isomerization at the individual cell level. We performed capillary electrophoresis (CE) with mass spectrometry (MS) detection to characterize the peptide content of single cells. Using complementary trapped ion mobility spectrometry (TIMS) separations, we measured the stereochemical configurations of three neuropeptide gene products derived from the pleurin precursor in individual neurons (N = 3) isolated from the central nervous system of Aplysia californica. An analysis of the resultant mobility profiles indicated >98% of the detectable pleurin-derived peptides exist as the nonisomerized, all-l forms in individual neuron cell bodies. However, we observed 44% of the Plrn2 peptide from the pleurin precursor was present as the isomerized, d-residue-containing form in the nerve tissue. These findings demonstrate an unusual distribution of isomerized peptides in A. californica and establish CE-TIMS MS as a powerful analytical tool for investigating peptide stereochemistry at the single cell level.

Advancing d-amino acid-containing peptide discovery in the metazoan.

The discovery of enzyme-derived d-amino acid-containing peptides (DAACPs) that have physiological importance in the metazoan challenges previous assumptions about the homochirality of animal proteins while simultaneously revealing new analytical challenges in the structural and functional characterization of peptides. Most known DAACPs have been identified though laborious activity-guided purification studies or by homology to previously identified DAACPs. Peptide characterization experiments are increasingly dominated by high throughput mass spectrometry-based peptidomics, with stereochemistry rarely considered due to the technical challenges of identifying l/d isomerization. This review discusses the prevalence of enzyme-derived DAACPs among animals and the physiological consequences of peptide isomerization. Also highlighted are the analytical methods that have been applied for structural characterization/discovery of DAACPs, including results of several recent studies using non-targeted discovery methods for revealing novel DAACPs, strongly suggesting that more DAACPs remain to be uncovered.

Bioinformatic and Reactivity-Based Discovery of Linaridins.

Linaridins are members of the ribosomally synthesized and post-translationally modified peptide (RiPP) family of natural products. Five linaridins have been reported, which are defined by the presence of dehydrobutyrine, a dehydrated, alkene-containing amino acid derived from threonine. This work describes the development of a linaridin-specific scoring module for Rapid ORF Description and Evaluation Online (RODEO), a genome-mining tool tailored toward RiPP discovery. Upon mining publicly accessible genomes available in the NCBI database, RODEO identified 561 (382 nonredundant) linaridin biosynthetic gene clusters. Linaridin BGCs with unique gene architectures and precursor sequences markedly different from previous predictions were uncovered during these efforts. To aid in data set validation, two new linaridins, pegvadin A and B, were detected through reactivity-based screening and isolated from Streptomyces noursei and Streptomyces auratus, respectively. Reactivity-based screening involves the use of a probe that chemoselectively modifies an organic functional group present in the natural product. The dehydrated amino acids present in linaridins as α/ß-unsaturated carbonyls were appropriate electrophiles for nucleophilic 1,4-addition using a thiol-functionalized probe. The data presented within significantly expand the number of predicted linaridin biosynthetic gene clusters and serve as a roadmap for future work in the area. The combination of bioinformatics and reactivity-based screening is a powerful approach to accelerate natural product discovery.

Differential Post-Translational Amino Acid Isomerization Found among Neuropeptides in Aplysia californica.

d-Amino acid-containing peptides (DAACPs) make up a class of post-translationally modified peptides in animals that play important roles as cell-to-cell signaling molecules. Despite the functional importance of l- to d-residue isomerization, little is known about its prevalence, mostly due to difficulties associated with detecting differences in peptide stereochemistry. Prior efforts to discover DAACPs have been largely focused on pursuing peptides based on homology to known DAACPs or DAACP-encoding precursors. Here, we used a combination of enzymatic screening, mass spectrometry, and chromatographic analysis to identify novel DAACPs in the central nervous system (CNS) of Aplysia californica. We identified five new DAACPs from the pleurin precursor and three DAACPs from previously uncharacterized proteins. In addition, two peptides from the pleurin precursor, Plrn2 and Plrn3, exist as DAACPs with the d-residue found at position 2 or 3. These differentially modified forms of Plrn2 and Plrn3 are located in specific regions of the animal's CNS. Plrn2 and Plrn3 appear to be the first animal DAACPs in which the d-residue is found at more than one position, and this suggests that l- to d-residue isomerization may be a more variable/dynamic modification than previously thought. Overall, this study demonstrates the utility of nontargeted DAACP discovery approaches for identifying new DAACPs and demonstrates that isomerization is prevalent throughout the CNS of A. californica.