Surveying the sequence diversity of model prebiotic peptides by mass spectrometry.

The rise of peptides with secondary structures and functions would have been a key step in the chemical evolution which led to life. As with modern biology, amino acid sequence would have been a primary determinant of peptide structure and activity in an origins-of-life scenario. It is a commonly held hypothesis that unique functional sequences would have emerged from a diverse soup of proto-peptides, yet there is a lack of experimental data in support of this. Whereas the majority of studies in the field focus on peptides containing only one or two types of amino acids, here we used modern mass spectrometry (MS)-based techniques to separate and sequence de novo proto-peptides containing broader combinations of prebiotically plausible monomers. Using a dry-wet environmental cycling protocol, hundreds of proto-peptide sequences were formed over a mere 4 d of reaction. Sequence homology diagrams were constructed to compare experimental and theoretical sequence spaces of tetrameric proto-peptides. MS-based analyses such as this will be increasingly necessary as origins-of-life researchers move toward systems-level investigations of prebiotic chemistry.

Compositional characterization of complex protopeptide libraries via triboelectric nanogenerator Orbitrap mass spectrometry.

RATIONALE: Understanding of the molecular processes that led to the first biomolecules on Earth is one of the key aspects of origins-of-life research. Depsipeptides, or polymers with mixed amide and ester backbones, have been proposed as plausible prebiotic precursors for peptide formation. Chemical characterization of depsipeptides in complex prebiotic-like mixtures should benefit from more efficient ion sources and ultrahigh-resolution mass spectrometry (UHR-MS) for elemental composition elucidation. METHODS: A sliding freestanding (SF) Triboelectric Nanogenerator (TENG) was coupled to glass nanoelectrospray emitters for the analysis of a depsipeptide library created using 11 amino acids and 3 alpha-hydroxy acids subjected to environmentally driven polymerization. The TENG nanoelectrospray ionization (nanoESI) source was coupled to an UHR Orbitrap mass spectrometer operated at 1,000,000 resolution for detecting depsipeptides and oligoesters in such libraries. Tandem mass spectrometry (MS/MS) experiments were performed on an Orbitrap Q-Exactive mass spectrometer. RESULTS: Our previous proteomics-like approach to depsipeptide library characterization showed the enormous complexity of these dynamic combinatorial systems. Here, direct infusion UHR-MS along with de novo sequencing enabled the identification of 524 sequences corresponding to 320 different depsipeptide compositions. Van Krevelen and mass defect diagrams enabled better visualization of the chemical diversity in these synthetic libraries. CONCLUSIONS: TENG nanoESI coupled to UHR-MS is a powerful method for depsipeptide library characterization in an origins-of-life context.

Structural Characterization of Methylenedianiline Regioisomers by Ion Mobility-Mass Spectrometry and Tandem Mass Spectrometry. 4. 3-Ring and 4-Ring Isomers.

Matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) is used to characterize methylenedianiline (MDA) 3-ring and 4-ring species. Building on our previous MALDI-MS 2-ring MDA isomer study, here we compare 3-ring and 4-ring electrospray ionization (ESI) and MALDI results. In ESI, 3-ring and 4-ring MDAs each form a single [M + H]+ parent ion. However, in MALDI, each MDA multimer forms three unique precursor ions: [M + H]+, [M•]+, and [M - H]+. In this study, 3-ring and 4-ring MDA precursors are characterized to identify the unique fragment ions formed and their respective fragmentation pathways. In addition to the three possible precursors, the 3-ring and 4-ring species are higher-order oligomer precursors in polyurethane (PUR) production and thus provide additional insight into the polymeric behavior of these PUR hard block precursors. The combination of ion mobility-mass spectrometry (IM - MS) and tandem mass spectrometry (MS/MS) allow the structural characterization of these larger MDA multimers.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of model prebiotic peptides: Optimization of sample preparation.

RATIONALE: Depsipeptides, or peptides with a mixture of amide and ester linkages, may have evolved into peptides on primordial Earth. Previous studies on depsipeptides utilized electrospray ionization ion mobility quadrupole time-of-flight (ESI-IM-QTOF) tandem mass spectrometry; such analysis was thorough yet time-consuming. Here, a complementary matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) approach was optimized for rapid characterization of depsipeptide length and monomer composition. METHODS: Depsipeptide mixtures of varying hydrophobicity were formed by subjecting aqueous mixtures of α-hydroxy acids and α-amino acids to evaporative cycles. Ester and amide content of depsipeptides was orthogonally confirmed using infrared spectroscopy. MALDI-TOF MS analysis was performed on a Voyager DE-STR in reflection geometry and positive ion mode. Optimization parameters included choice of matrix, sample solvent, matrix-to-analyte ratio, and ionization additives. RESULTS: It was determined that evaporated depsipeptide samples should be mixed with 2,5-dihydroxybenzoic acid (DHB) matrix in order to detect the highest number of unique signals. Low matrix-to-analyte ratios were found to generate higher quality spectra, likely due to a combination of matrix suppression and improved co-crystallization. Using this optimized protocol, a new depsipeptide mixture was characterized. CONCLUSIONS: Understanding the diversity and chemical evolution of proto-peptides is of interest to origins-of-life research. Here, we have demonstrated MALDI-TOF MS can be used to rapidly screen the length and monomer composition of model prebiotic peptides containing a mixture of ester and amide backbone linkages.

Synthesis of ß-Peptide Standards for Use in Model Prebiotic Reactions.

A one-pot method was developed for the preparation of a series of ß-alanine standards of moderate size (2 to ≥12 residues) for studies concerning the prebiotic origins of peptides. The one-pot synthesis involved two sequential reactions: (1) dry-down self-condensation of ß-alanine methyl ester, yielding ß-alanine peptide methyl ester oligomers, and (2) subsequent hydrolysis of ß-alanine peptide methyl ester oligomers, producing a series of ß-alanine peptide standards. These standards were then spiked into a model prebiotic product mixture to confirm by HPLC the formation of ß-alanine peptides under plausible reaction conditions. The simplicity of this approach suggests it can be used to prepare a variety of ß-peptide standards for investigating differences between α- and ß-peptides in the context of prebiotic chemistry.

Structural Characterization of Methylenedianiline Regioisomers by Ion Mobility-Mass Spectrometry, Tandem Mass Spectrometry, and Computational Strategies. 3. MALDI Spectra of 2-Ring Isomers.

Characterization of methylenedianiline (MDA) 2-ring isomers (2,2'-, 2,4'-, and 4,4'-MDA) is reported using matrix assisted laser desorption/ionization-mass spectrometry (MALDI-MS), a common technique used for characterizing synthetic polymers. MDA is a precursor to methylene diphenyl diisocyanate (MDI), a hard block component in polyurethane (PUR) synthesis. This work focuses on comparing MALDI results to those of our previous electrospray ionization-mass spectrometry (ESI-MS) studies. In ESI, 2-ring MDA isomers formed single unique [M + H]+ (199 Da) parent ions, whereas in MALDI each isomer shows significant formation of three precursor ions: [M - H]+ = 197 Da, [M•]+ = 198 Da, and [M + H]+ = 199 Da. Structures and schemes are proposed for the MALDI fragment ions associated with each precursor ion. Ion mobility-mass spectrometry (IM-MS), tandem mass spectrometry (MS/MS), and computational methods were all critical in determining the structures for both precursor and fragment ions as well as the fragmentation mechanisms. The present study indicates that the [M - H]+ and [M•]+ ions are formed by the MALDI process, explaining why they were not observed with ESI.

Structural Characterization of Methylenedianiline Regioisomers by Ion Mobility-Mass Spectrometry, Tandem Mass Spectrometry, and Computational Strategies. 2. Electrospray Spectra of 3-Ring and 4-Ring Isomers.

Building on results from our previous study of 2-ring methylenedianiline (MDA), a combined mass spectrometry approach utilizing ion mobility-mass spectrometry (IM-MS) and tandem mass spectrometry (MS/MS) coupled with computational methods enables the structural characterization of purified 3-ring and 4-ring MDA regioisomers in this current study. The preferred site of protonation for the 3-ring and 4-ring MDA was determined to be on the amino groups. Additionally, the location of the protonated amine along the MDA multimer was found to influence the gas phase stability of these molecules. Fragmentation mechanisms similar to the 2-ring MDA species were observed for both the 3-ring and 4-ring MDA. The structural characterization of 3-ring and 4-ring MDA isomers using modern MS techniques may aid polyurethane synthesis by the characterization of industrial grade MDA, multimeric MDA species, and methylene diphenyl diisocyanate (MDI) mixtures.

Collision cross section calibrants for negative ion mode traveling wave ion mobility-mass spectrometry.

Unlike traditional drift-tube ion mobility-mass spectrometry, traveling-wave ion mobility-mass spectrometry typically requires calibration in order to generate collision cross section (CCS) values. Although this has received a significant amount of attention for positive-ion mode analysis, little attention has been paid for CCS calibration in negative ion mode. Here, we provide drift-tube CCS values for [M - H](-) ions of two calibrant series, polyalanine and polymalic acid, and evaluate both types of calibrants in terms of the accuracy and precision of the traveling-wave ion mobility CCS values that they produce.

Ester-Mediated Amide Bond Formation Driven by Wet-Dry Cycles: A Possible Path to Polypeptides on the Prebiotic Earth.

Although it is generally accepted that amino acids were present on the prebiotic Earth, the mechanism by which α-amino acids were condensed into polypeptides before the emergence of enzymes remains unsolved. Here, we demonstrate a prebiotically plausible mechanism for peptide (amide) bond formation that is enabled by α-hydroxy acids, which were likely present along with amino acids on the early Earth. Together, α-hydroxy acids and α-amino acids form depsipeptides-oligomers with a combination of ester and amide linkages-in model prebiotic reactions that are driven by wet-cool/dry-hot cycles. Through a combination of ester-amide bond exchange and ester bond hydrolysis, depsipeptides are enriched with amino acids over time. These results support a long-standing hypothesis that peptides might have arisen from ester-based precursors.

Phosphorylation of serine 106 in Asef2 regulates cell migration and adhesion turnover.

Asef2, a 652-amino acid protein, is a guanine nucleotide exchange factor (GEF) that regulates cell migration and other processes via activation of Rho family GTPases, including Rac. Binding of the tumor suppressor adenomatous polyposis coli (APC) to Asef2 is known to induce its GEF activity; however, little is currently known about other modes of Asef2 regulation. Here, we investigated the role of phosphorylation in regulating Asef2 activity and function. Using high-resolution mass spectrometry (MS) and tandem mass spectrometry (MS/MS), we obtained complete coverage of all phosphorylatable residues and identified six phosphorylation sites. One of these, serine 106 (S106), was particularly intriguing as a potential regulator of Asef2 activity because of its location within the APC-binding domain. Interestingly, mutation of this serine to alanine (S106A), a non-phosphorylatable analogue, greatly diminished the ability of Asef2 to activate Rac, while a phosphomimetic mutation (serine to aspartic acid, S106D) enhanced Rac activation. Furthermore, expression of these mutants in HT1080 cells demonstrated that phosphorylation of S106 is critical for Asef2-promoted migration and for cell-matrix adhesion assembly and disassembly (adhesion turnover), which is a process that facilitates efficient migration. Collectively, our results show that phosphorylation of S106 modulates Asef2 GEF activity and Asef2-mediated cell migration and adhesion turnover.

Structural characterization of methylenedianiline regioisomers by ion mobility-mass spectrometry, tandem mass spectrometry, and computational strategies: I. Electrospray spectra of 2-ring isomers.

Purified methylenedianiline (MDA) regioisomers were structurally characterized and differentiated using tandem mass spectrometry (MS/MS), ion mobility-mass spectrometry (IM-MS), and IM-MS/MS in conjunction with computational methods. It was determined that protonation sites on the isomers can vary depending on the position of amino groups, and the resulting protonation sites play a role in the gas-phase stability of the isomer. We also observed differences in the relative distributions of protonated conformations depending on experimental conditions and instrumentation, which is consistent with previous studies on aniline in the gas phase. This work demonstrates the utility of a multifaceted approach for the study of isobaric species and elucidates why previous MDA studies may have been unable to detect and/or differentiate certain isomers. Such analysis may prove useful in the characterization of larger MDA multimeric species, industrial MDA mixtures, and methylene diphenyl diisocyanate (MDI) mixtures used in polyurethane synthesis.

Qualitative Monitoring of Proto-Peptide Condensation by Differential FTIR Spectroscopy.

Condensation processes such as wet-dry cycling are thought to have played significant roles in the emergence of proto-peptides. Here, we describe a simple and low-cost method, differential Fourier transform infrared (FTIR) spectroscopy, for qualitative analysis of peptide condensation products in model primordial reactions. We optimize differential FTIR for depsipeptides and apply this method to investigate their polymerization in the presence of extraterrestrial dust simulants.

Semitransparent nanostructured films for imaging mass spectrometry and optical microscopy.

Semitransparent porous silicon substrates have been developed for pairing nanostructure-initiator mass spectrometry (NIMS) imaging with traditional optical-based microscopy techniques. Substrates were optimized to generate the largest NIMS signal while maintaining sufficient transparency to allow visible light to pass through for optical microscopy. Using these substrates, both phase-contrast and NIMS images of phospholipids from a scratch-wounded cell monolayer were obtained. NIMS images were generated using a spatial resolution of 14 µm. Coupled with further improvements in spatial resolution, this approach may allow for the localization of intact biological molecules within cells without the need for labeling.

Differential Oligomerization of Alpha versus Beta Amino Acids and Hydroxy Acids in Abiotic Proto-Peptide Synthesis Reactions.

The origin of biopolymers is a central question in origins of life research. In extant life, proteins are coded linear polymers made of a fixed set of twenty alpha-L-amino acids. It is likely that the prebiotic forerunners of proteins, or protopeptides, were more heterogenous polymers with a greater diversity of building blocks and linkage stereochemistry. To investigate a possible chemical selection for alpha versus beta amino acids in abiotic polymerization reactions, we subjected mixtures of alpha and beta hydroxy and amino acids to single-step dry-down or wet-dry cycling conditions. The resulting model protopeptide mixtures were analyzed by a variety of analytical techniques, including mass spectrometry and NMR spectroscopy. We observed that amino acids typically exhibited a higher extent of polymerization in reactions that also contained alpha hydroxy acids over beta hydroxy acids, whereas the extent of polymerization by beta amino acids was higher compared to their alpha amino acid analogs. Our results suggest that a variety of heterogenous protopeptide backbones existed during the prebiotic epoch, and that selection towards alpha backbones occurred later as a result of polymer evolution.

Prebiotic condensation through wet-dry cycling regulated by deliquescence.

Wet-dry cycling is widely regarded as a means of driving condensation reactions under prebiotic conditions to generate mixtures of prospective biopolymers. A criticism of this model is its reliance on unpredictable rehydration events, like rainstorms. Here, we report the ability of deliquescent minerals to mediate the oligomerization of glycine during iterative wet-dry cycles. The reaction mixtures evaporate to dryness at high temperatures and spontaneously reacquire water vapor to form aqueous solutions at low temperatures. Deliquescent mixtures can foster yields of oligomerization over ten-fold higher than non-deliquescent controls. The deliquescent mixtures tightly regulate their moisture content, which is crucial, as too little water precludes dissolution of the reactants while too much water favors hydrolysis over condensation. The model also suggests a potential reason why life evolved to favor the enrichment of potassium: so living systems could acquire and retain sufficient water to serve as a solvent for biochemical reactions.

Profiling and Imaging Ion Mobility-Mass Spectrometry Analysis of Cholesterol and 7-Dehydrocholesterol in Cells Via Sputtered Silver MALDI.

Profiling and imaging of cholesterol and its precursors by mass spectrometry (MS) are important in a number of cholesterol biosynthesis disorders, such as in Smith-Lemli-Opitz syndrome (SLOS), where 7-dehydrocholesterol (7-DHC) is accumulated in affected individuals. SLOS is caused by defects in the enzyme that reduces 7-DHC to cholesterol. However, analysis of sterols is challenging because these hydrophobic olefins are difficult to ionize for MS detection. We report here sputtered silver matrix-assisted laser desorption/ionization (MALDI)-ion mobility-MS (IM-MS) analysis of cholesterol and 7-DHC. In comparison with liquid-based AgNO3 and colloidal Ag nanoparticle (AgNP), sputtered silver NP (10-25 nm) provided the lowest limits-of-detection based on the silver coordinated [cholesterol + Ag](+) and [7-DHC + Ag](+) signals while minimizing dehydrogenation products ([M + Ag-2H](+)). When analyzing human fibroblasts that were directly grown on poly-L-lysine-coated ITO glass plates with this technique, in situ, the 7-DHC/cholesterol ratios for both control and SLOS human fibroblasts are readily obtained. The m/z of 491 (specific for [7-DHC + (107)Ag](+)) and 495 (specific for [cholesterol + (109)Ag](+)) were subsequently imaged using MALDI-IM-MS. MS images were co-registered with optical images of the cells for metabolic ratio determination. From these comparisons, ratios of 7-DHC/cholesterol for SLOS human fibroblasts are distinctly higher than in control human fibroblasts. Thus, this strategy demonstrates the utility for diagnosing/assaying the severity of cholesterol biosynthesis disorders in vitro.