The next decade of protein structure.

With recent dramatic advances in various techniques used for protein structure research, we asked researchers to comment on the next exciting questions for the field and about how these techniques will advance our knowledge not only about proteins but also about human health and diseases.

ROS-dependent S-palmitoylation activates cleaved and intact gasdermin D.

Gasdermin D (GSDMD) is the common effector for cytokine secretion and pyroptosis downstream of inflammasome activation and was previously shown to form large transmembrane pores after cleavage by inflammatory caspases to generate the GSDMD N-terminal domain (GSDMD-NT)1-10. Here we report that GSDMD Cys191 is S-palmitoylated and that palmitoylation is required for pore formation. S-palmitoylation, which does not affect GSDMD cleavage, is augmented by mitochondria-generated reactive oxygen species (ROS). Cleavage-deficient GSDMD (D275A) is also palmitoylated after inflammasome stimulation or treatment with ROS activators and causes pyroptosis, although less efficiently than palmitoylated GSDMD-NT. Palmitoylated, but not unpalmitoylated, full-length GSDMD induces liposome leakage and forms a pore similar in structure to GSDMD-NT pores shown by cryogenic electron microscopy. ZDHHC5 and ZDHHC9 are the major palmitoyltransferases that mediate GSDMD palmitoylation, and their expression is upregulated by inflammasome activation and ROS. The other human gasdermins are also palmitoylated at their N termini. These data challenge the concept that cleavage is the only trigger for GSDMD activation. They suggest that reversible palmitoylation is a checkpoint for pore formation by both GSDMD-NT and intact GSDMD that functions as a general switch for the activation of this pore-forming family.

Detergents with Scalable Properties Identify Noncanonical Lipopolysaccharide Binding to Bacterial Inner Membrane Proteins.

Lipopolysaccharide (LPS) is vital for maintaining the outer membrane barrier in Gram-negative bacteria. LPS is also frequently obtained in complex with the inner membrane proteins after detergent purification. The question of whether or not LPS binding to inner membrane proteins not involved in outer membrane biogenesis reflects native lipid environments remains unclear. Here, we leverage the control of the hydrophilic-lipophilic balance and packing parameter concepts to chemically tune detergents that can be used to qualitatively differentiate the degree to which proteins copurify with phospholipids (PLs) and/or LPS. Given the scalable properties of these detergents, we demonstrate a detergent fine-tuning that enables the facile investigation of intact proteins and their complexes with lipids by native mass spectrometry (nMS). We conclude that LPS, a lipid that is believed to be important for outer membranes, can also affect the activity of membrane proteins that are currently not assigned to be involved in outer membrane biogenesis. Our results deliver a scalable detergent chemistry for a streamlined biophysical characterization of protein-lipid interactions, provide a rationale for the high affinity of LPS-protein binding, and identify noncanonical associations between LPS and inner membrane proteins with relevance for membrane biology and antibiotic research.

ProSight Native: Defining Protein Complex Composition from Native Top-Down Mass Spectrometry Data.

Native mass spectrometry has recently moved alongside traditional structural biology techniques in its ability to provide clear insights into the composition of protein complexes. However, to date, limited software tools are available for the comprehensive analysis of native mass spectrometry data on protein complexes, particularly for experiments aimed at elucidating the composition of an intact protein complex. Here, we introduce ProSight Native as a start-to-finish informatics platform for analyzing native protein and protein complex data. Combining mass determination via spectral deconvolution with a top-down database search and stoichiometry calculations, ProSight Native can determine the complete composition of protein complexes. To demonstrate its features, we used ProSight Native to successfully determine the composition of the homotetrameric membrane complex Aquaporin Z. We also revisited previously published spectra and were able to decipher the composition of a heterodimer complex bound with two noncovalently associated ligands. In addition to determining complex composition, we developed new tools in the software for validating native mass spectrometry fragment ions and mapping top-down fragmentation data onto three-dimensional protein structures. Taken together, ProSight Native will reduce the informatics burden on the growing field of native mass spectrometry, enabling the technology to further its reach.

Uncovering the Role of N-Glycan Occupancy on the Cooperative Assembly of Spike and Angiotensin Converting Enzyme 2 Complexes: Insights from Glycoengineering and Native Mass Spectrometry.

Interactions between the SARS-CoV-2 Spike protein and ACE2 are one of the most scrutinized reactions of our time. Yet, questions remain as to the impact of glycans on mediating ACE2 dimerization and downstream interactions with Spike. Here, we address these unanswered questions by combining a glycoengineering strategy with high-resolution native mass spectrometry (MS) to investigate the impact of N-glycan occupancy on the assembly of multiple Spike-ACE2 complexes. We confirmed that intact Spike trimers have all 66 N-linked sites occupied. For monomeric ACE2, all seven N-linked glycan sites are occupied to various degrees; six sites have >90% occupancy, while the seventh site (Asn690) is only partially occupied (∼30%). By resolving the glycoforms on ACE2, we deciphered the influence of each N-glycan on ACE2 dimerization. Unexpectedly, we found that Asn432 plays a role in mediating dimerization, a result confirmed by site-directed mutagenesis. We also found that glycosylated dimeric ACE2 and Spike trimers form complexes with multiple stoichiometries (Spike-ACE2 and Spike2-ACE2) with dissociation constants (Kds) of ∼500 and <100 nM, respectively. Comparing these values indicates that positive cooperativity may drive ACE2 dimers to complex with multiple Spike trimers. Overall, our results show that occupancy has a key regulatory role in mediating interactions between ACE2 dimers and Spike trimers. More generally, since soluble ACE2 (sACE2) retains an intact SARS-CoV-2 interaction site, the importance of glycosylation in ACE2 dimerization and the propensity for Spike and ACE2 to assemble into higher oligomers are molecular details important for developing strategies for neutralizing the virus.

Rationalizing the Optimization of Detergents for Membrane Protein Purification.

Membrane protein purification by means of detergents is key to isolating membrane-bound therapeutic targets. The role of the detergent structure in this process, however, is not well understood. Detergents are optimized empirically, leading to failed preparations, and thereby raising costs. Here we evaluate the utility of the hydrophilic-lipophilic balance (HLB) concept, which was introduced by Griffin in 1949, for guiding the optimization of the hydrophobic tail in first-generation, dendritic oligoglycerol detergents ([G1] OGDs). Our findings deliver qualitative HLB guidelines for rationalizing the optimization of detergents. Moreover, [G1] OGDs exhibit strongly delipidating properties, regardless of the structure of the hydrophobic tail, which delivers a methodological enabling step for investigating binding strengths of endogenous lipids and their role for membrane protein oligomerization. Our findings will facilitate the analysis of challenging drug targets in the future.

Tautomerization of H+KPGG: Entropic Consequences of Strong Hydrogen-Bond Networks in Peptides.

Ion mobility spectrometry-mass spectrometry and quantum chemical calculations are used to determine the structures and stabilities of the singly protonated peptide H+KPGG. The two peaks making up the IMS distribution are shown to be tautomers differing by the location of the extra proton on either the lysine side chain or the N-terminus. The lysine-protonated tautomer is strongly preferred entropically while being disfavored in terms of the electronic energy and enthalpy. This relationship is shown, through comparison of all low-lying conformers of both tautomers, to be related to the strong hydrogen-bond network of the N-terminally protonated tautomer. A general relationship is demonstrated wherein stronger cross-peptide hydrogen-bond networks result in entropically disfavored conformers. Further effects of the H+KPGG hydrogen-bond network are probed by computationally examining singly and doubly methylated analogues. These results demonstrate the importance of the entropic consequences of hydrogen bonds to peptide stability as well as techniques for perturbing the hydrogen-bond network and folding preferences of peptides via minimal chemical modification.

Variation of CI-2 Conformers upon Addition of Methanol to Water: An IMS-MS-Based Thermodynamic Analysis.

Chymotrypsin inhibitor 2 (CI-2) is a well-studied, textbook example of a cooperative, two-state, native ↔ denatured folding transition. A recent hybrid ion mobility spectrometry (IMS)/mass spectrometry (MS) thermal denaturation study of CI-2 (the well-studied truncated 64-residue model) in water reported evidence that this two-state transition involves numerous (∼41) unique native and non-native (denatured) solution conformations. The characterization of so many, often low-abundance, states is possible because of the very high dynamic range of IMS-MS measurements of ionic species that are produced upon electrospraying CI-2 solutions from a variable temperature electrospray ionization source. A thermodynamic analysis of these states revealed large changes in enthalpy (ΔH) and entropy (ΔS) at different temperatures, and it was suggested that such variation might arise because of temperature-dependent conformational changes of the protein in response to changes in the conformational entropy and the dielectric permeability of water, which drops from a value of ε ∼ 79 at 24 °C to ∼ 60 at 82 °C. Herein, we examine how adding methanol to water influences the distributions of CI-2 conformers and their ensuing stabilities. The dielectric constant of a 60:40 water:methanol (MeOH) drops from ε ∼ 60 at 24 °C to ∼ 51 at 64 °C. Although the same set of conformers observed in water appears to be present in 60:40 water:MeOH, the abundance of each is substantially altered by the presence of methanol. Relative free energy values (ΔG) and thermodynamic values [ΔH and ΔS and heat capacities (ΔCp)] are derived from a Gibbs-Helmholtz analysis. A comparison of these data from water and water:MeOH systems allows rare insight into how variations in solvation and temperature affect many-state protein equilibria. While these studies confirm that variations in solvent dielectric constant with temperature affect the distributions of conformers that are observed, our findings suggest that other solvent differences may also affect abundances.

Infrared Multiphoton Dissociation Enables Top-Down Characterization of Membrane Protein Complexes and G Protein-Coupled Receptors.

Membrane proteins are challenging to analyze by native mass spectrometry (MS) as their hydrophobic nature typically requires stabilization in detergent micelles that are removed prior to analysis via collisional activation. There is however a practical limit to the amount of energy which can be applied, which often precludes subsequent characterization by top-down MS. To overcome this barrier, we have applied a modified Orbitrap Eclipse Tribrid mass spectrometer coupled to an infrared laser within a high-pressure linear ion trap. We show how tuning the intensity and time of incident photons enables liberation of membrane proteins from detergent micelles. Specifically, we relate the ease of micelle removal to the infrared absorption of detergents in both condensed and gas phases. Top-down MS via infrared multiphoton dissociation (IRMPD), results in good sequence coverage enabling unambiguous identification of membrane proteins and their complexes. By contrasting and comparing the fragmentation patterns of the ammonia channel with two class A GPCRs, we identify successive cleavage of adjacent amino acids within transmembrane domains. Using gas-phase molecular dynamics simulations, we show that areas prone to fragmentation maintain aspects of protein structure at increasing temperatures. Altogether, we propose a rationale to explain why and where in the protein fragment ions are generated.

Heterogeneity of Glycan Processing on Trimeric SARS-CoV-2 Spike Protein Revealed by Charge Detection Mass Spectrometry.

The heterogeneity associated with glycosylation of the 66 N-glycan sites on the protein trimer making up the spike (S) region of the SARS-CoV-2 virus has been assessed by charge detection mass spectrometry (CDMS). CDMS allows simultaneous measurement of the mass-to-charge ratio and charge of individual ions, so that mass distributions can be determined for highly heterogeneous proteins such as the heavily glycosylated S protein trimer. The CDMS results are compared to recent glycoproteomics studies of the structure and abundance of glycans at specific sites. Interestingly, average glycan masses determined by "top-down" CDMS measurements are 35-47% larger than those obtained from the "bottom-up" glycoproteomics studies, suggesting that the glycoproteomic measurements underestimated the abundances of larger, more-complex glycans. Moreover, the distribution of glycan masses determined by CDMS is much broader than the distribution expected from the glycoproteomics studies, assuming that glycan processing on each trimer is not correlated. The breadth of the glycan mass distribution therefore indicates heterogeneity in the extent of glycan processing of the S protein trimers, with some trimers being much more heavily processed than others. This heterogeneity may have evolved as a way of further confounding the host's immune system.

Thermal Analysis of a Mixture of Ribosomal Proteins by vT-ESI-MS: Toward a Parallel Approach for Characterizing the Stabilitome.

The thermal stabilities of endogenous, intact proteins and protein assemblies in complex mixtures were characterized in parallel by means of variable-temperature electrospray ionization coupled to mass spectrometry (vT-ESI-MS). The method is demonstrated by directly measuring the melting transitions of seven proteins from a mixture of proteins derived from ribosomes. A proof-of-concept measurement of a fraction of an Escherichia coli lysate is provided to extend this approach to characterize the thermal stability of a proteome. As the solution temperature is increased, proteins and protein complexes undergo structural and organizational transitions; for each species, the folded ↔ unfolded and assembled ↔ disassembled populations are monitored based on changes in vT-ESI-MS charge state distributions and masses. The robustness of the approach illustrates a step toward the proteome-wide characterization of thermal stabilities and structural transitions-the stabilitome.

Comparison of gaseous ubiquitin ion structures obtained from a solid and solution matrix using ion mobility spectrometry/mass spectrometry.

RATIONALE: Examining surface protein conformations, and especially achieving this with spatial resolution, is an important goal. The recently discovered ionization processes offer spatial-resolution measurements similar to matrix-assisted laser desorption/ionization (MALDI) and produce charge states similar to electrospray ionization (ESI) extending higher-mass protein applications directly from surfaces on high-performance mass spectrometers. Studying a well-interrogated protein by ion mobility spectrometry-mass spectrometry (IMS-MS) to access effects on structures using a solid vs. solvent matrix may provide insights. METHODS: Ubiquitin was studied by IMS-MS using new ionization processes with commercial and homebuilt ion sources and instruments (Waters SYNAPT G2(S)) and homebuilt 2 m drift-tube instrument; MS™ sources). Mass-to-charge and drift-time (td )-measurements are compared for ubiquitin ions obtained by inlet and vacuum ionization using laserspray ionization (LSI), matrix- (MAI) and solvent-assisted ionization (SAI), respectively, and compared with those from ESI under conditions that are most comparable. RESULTS: Using the same solution conditions with SYNAPT G2(S) instruments, td -distributions of various ubiquitin charge states from MAI, LSI, and SAI are similar to those from ESI using a variety of solvents, matrices, extraction voltages, a laser, and temperature only, showing subtle differences in more compact features within the elongated distribution of structures. However, on a homebuilt drift-tube instrument, within the elongated distribution of structures, both similar and different td -distributions are observed for ubiquitin ions obtained by MAI and ESI. MAI-generated ions are frequently narrower in their td -distributions. CONCLUSIONS: Direct comparisons between ESI and the new ionization methods operational directly from surfaces suggest that the protein in its solution structure prior to exposure to the ionization event is either captured (frozen out) at the time of crystallization, or that the protein in the solid matrix is associated with sufficient solvent to maintain the solution structure, or, alternatively, that the observed structures are those related to what occurs in the gas phase with ESI- or MAI-generated ions and not with the solution structures.

An overview of biological applications and fundamentals of new inlet and vacuum ionization technologies.

RATIONALE: The developments of new ionization technologies based on processes previously unknown to mass spectrometry (MS) have gained significant momentum. Herein we address the importance of understanding these unique ionization processes, demonstrate the new capabilities currently unmet by other methods, and outline their considerable analytical potential. METHODS: The inlet and vacuum ionization methods of solvent-assisted ionization (SAI), matrix-assisted ionization (MAI), and laserspray ionization can be used with commercial and dedicated ion sources producing ions from atmospheric or vacuum conditions for analyses of a variety of materials including drugs, lipids, and proteins introduced from well plates, pipet tips and plate surfaces with and without a laser using solid or solvent matrices. Mass spectrometers from various vendors are employed. RESULTS: Results are presented highlighting strengths relative to ionization methods of electrospray ionization (ESI) and matrix-assisted laser desorption/ionization. We demonstrate the utility of multi-ionization platforms encompassing MAI, SAI, and ESI and enabling detection of what otherwise is missed, especially when directly analyzing mixtures. Unmatched robustness is achieved with dedicated vacuum MAI sources with mechanical introduction of the sample to the sub-atmospheric pressure (vacuum MAI). Simplicity and use of a wide array of matrices are attained using a conduit (inlet ionization), preferably heated, with sample introduction from atmospheric pressure. Tissue, whole blood, urine (including mouse, chicken, and human origin), bacteria strains and chemical on-probe reactions are analyzed directly and, especially in the case of vacuum ionization, without concern of carryover or instrument contamination. CONCLUSIONS: Examples are provided highlighting the exceptional analytical capabilities associated with the novel ionization processes in MS that reduce operational complexity while increasing speed and robustness, achieving mass spectra with low background for improved sensitivity, suggesting the potential of this simple ionization technology to drive MS into areas currently underserved, such as clinical and medical applications.

Evidence for Many Unique Solution Structures for Chymotrypsin Inhibitor 2: A Thermodynamic Perspective Derived from vT-ESI-IMS-MS Measurements.

Chymotrypsin inhibitor 2 (CI-2) is a classic model for two-state cooperative protein folding and is one of the most extensively studied systems. Alan Fersht, a pioneer in the field of structural biology, has studied the wild-type (wt) and over 100 mutant forms of CI-2 with traditional analytical and biochemical techniques. Here, we examine wt CI-2 and three mutant forms (A16G, K11A, L32A) to demonstrate the utility of variable-temperature (vT) electrospray ionization (ESI) paired with ion mobility spectrometry (IMS) and mass spectrometry (MS) to map the free energy folding landscape. As the solution temperature is increased, the abundance of each of the six ESI charge states for wt CI-2 and each mutant is found to vary independently. These results require that at least six unique types of CI-2 solution conformers are present. Ion mobility analysis reveals that within each charge state there are additional conformers having distinct solution temperature profiles. A model of the data at ∼30 different temperatures for all four systems suggests the presence of 41 unique CI-2 solution conformations. A thermodynamic analysis of this system yields values of ΔCp as well as ΔG, ΔH, and ΔS for each state at every temperature studied. Detailed energy landscapes derived from these data provide a rare glimpse into Anfinsen's thermodynamic hypothesis and the process of thermal denaturation, normally thought of as a cooperative two-state transition involving the native state and unstructured denatured species. Specifically, as the temperature is varied, the entropies and enthalpies of different conformers undergo dramatic changes in magnitude and relative order to maintain the delicate balance associated with equilibrium.

Melting of Hemoglobin in Native Solutions as measured by IMS-MS.

Thermally induced structural transitions of the quaternary structure of the hemoglobin tetramer (human) in aqueous solution (150 mM ammonium acetate) were investigated using a variable temperature electrospray ionization (vt-ESI) technique in combination with ion mobility spectrometry (IMS) and mass spectrometry (MS) measurements. At low solution temperatures (28 to ∼40 °C), a heterotetrameric (α2ß2) complex is the most abundant species that is observed. When the solution temperature is increased, this assembly dissociates into heterodimers (holo αß forms) before ultimately forming insoluble aggregates at higher temperatures (>60 °C). In addition to the holo αß forms, a small population of αß dimers containing only a single heme ligand and having a dioxidation modification mapping to the ß subunit are observed. The oxidized heterodimers are less stable than the unmodified holo-heterodimer. The Cys93 residue of the ß subunit is the primary site of dioxidation. The close proximity of this post translational modification to both the αß subunit interface and the heme binding site suggests that this modification is coupled to the loss of the heme and decreased protein stability. Changes in the charge state and collision cross sections of these species indicate that the tetramers and dimers favor less compact structures at elevated temperatures (prior to temperatures where dissociation dominates).

New mass spectrometry concepts for characterization of synthetic polymers and additives.

RATIONALE: New ionization processes have been developed for biological mass spectrometry (MS) in which the matrix lifts the nonvolatile analyte into the gas phase as ions without any additional energy input. We rationalized that additional fundamental knowledge is needed to assess analytical utility for the field of synthetic polymers and additives. METHODS: Different mass spectrometers (Thermo Orbitrap (Q-)Exactive (Focus); Waters SYNAPT G2(S)) were employed. The formation of multiply charged polymer ions upon exposure of the matrix/analyte(/salt) sample to sub-atmospheric pressure directly from the solid state and surfaces facilitates the use of advanced mass spectrometers for detection of polymeric materials including consumer products (e.g., gum). RESULTS: Astonishingly, using nothing more than a small molecule matrix compound (e.g., 2-methyl-2-nitropropane-1,3-diol or 3-nitrobenzonitrile) and a salt (e.g., mono- or divalent cation(s)), such samples upon exposure to sub-atmospheric pressure transfer nonvolatile polymers and nonvolatile salts into the gas phase as multiply charged ions. These successes contradict the conventional understanding of ionization in MS, because can nonvolatile polymers be lifted in the gas phase as ions not only by as little as a volatile matrix but also by the salt required for ionizing the analyte through noncovalent metal cation adduction(s). Prototype vacuum matrix-assisted ionization (vMAI) and automated sources using a contactless approach are demonstrated for direct analyses of synthetic polymers and plasticizers, minimizing the risk of contamination using direct sample introduction into the mass spectrometer vacuum. CONCLUSIONS: Direct ionization methods from surfaces without the need of high voltage, a laser, or even applied heat are demonstrated for characterization of detailed materials using (ultra)high-resolution and accurate mass measurements enabled by the multiply charged ions extending the mass range of high-performance mass spectrometers and use of a split probe sample introduction device. Our vision is that, with further development of fundamentals and dedicated sources, both spatial- and temporal-resolution measurements are within reach if sensitivity is addressed for decreasing sample-size measurements.

Allosteric Inhibition of the SARS-CoV-2 Main Protease: Insights from Mass Spectrometry Based Assays*.

The SARS-CoV-2 main protease (Mpro ) cleaves along the two viral polypeptides to release non-structural proteins required for viral replication. MPro is an attractive target for antiviral therapies to combat the coronavirus-2019 disease. Here, we used native mass spectrometry to characterize the functional unit of Mpro . Analysis of the monomer/dimer equilibria reveals a dissociation constant of Kd =0.14±0.03 µM, indicating MPro has a strong preference to dimerize in solution. We characterized substrate turnover rates by following temporal changes in the enzyme-substrate complexes, and screened small molecules, that bind distant from the active site, for their ability to modulate activity. These compounds, including one proposed to disrupt the dimer, slow the rate of substrate processing by ≈35 %. This information, together with analysis of the x-ray crystal structures, provides a starting point for the development of more potent molecules that allosterically regulate MPro activity.

Variable-Temperature ESI-IMS-MS Analysis of Myohemerythrin Reveals Ligand Losses, Unfolding, and a Non-Native Disulfide Bond.

Variable-temperature electrospray ionization combined with ion mobility spectrometry (IMS) and mass spectrometry (MS) techniques are used to monitor structural transitions of the protein myohemerythrin from peanut worm in aqueous ammonium acetate solutions from ∼15 to 92 °C. At physiological temperatures, myohemerythrin favors a four-helix bundle motif and has a diiron oxo cofactor that binds oxygen. As the solution temperature is increased from ∼15 to 35 °C, some bound oxygen dissociates; at ∼66 °C, the cofactor dissociates to produce populations of both folded and unfolded apoprotein. At higher temperatures (∼85 °C and above), the IMS-MS spectrum indicates that the folded apoprotein dominates, and provides evidence for stabilization of the structure by formation of a non-native disulfide bond. In total, we find evidence for 18 unique forms of myohemerythrin as well as information about the structures and stabilities of these states. The high-fidelity of IMS-MS techniques provides a means of examining the stabilities of individual components of complex mixtures that are inaccessible by traditional calorimetric and spectroscopic methods.

Solution thermochemistry of concanavalin A tetramer conformers measured by variable-temperature ESI-IMS-MS.

Variable-temperature nano-electrospray ionization coupled with ion mobility spectrometry-mass spectrometry is used to investigate the thermal denaturation of the tetrameric protein concanavalin A. As the solution temperature is increased, changes in mass spectra and collision cross section distributions provide evidence for discrete structural changes that occur at temperatures that are ~40 to 50 degrees below the temperature required for tetramer dissociation. The subtle structural changes are associated with four distinct tetramer conformations with unique melting temperatures. Gibbs-Helmholtz analysis of the free energies determined with respect to the most abundant "native" state yields heat capacities of ΔCp = 1.6 ± 0.3, -2.2 ± 0.4, and -2.9 ± 1.6 kJ·K-1·mol-1, and temperature dependent enthalpies and entropies for the three non-native conformations. Analysis of the thermochemistry indicates that the high-temperature products are entropically stable until the threshold for tetramer dissociation, and changes in heat capacity are consistent with increases in solvation of polar residues. Our findings suggest these high-temperature non-native states result from an increase in disorder at surface exposed regions. Such studies provide valuable insight towards the structural details of non-native states.

Monitoring the stabilities of a mixture of peptides by mass-spectrometry-based techniques.

Biomolecular degradation plays a key role in proteostasis. Typically, proteolytic enzymes degrade proteins into smaller peptides by breaking amino acid bonds between specific residues. Cleavage around proline residues is often missed and requires highly specific enzymes for peptide processing due to the cyclic proline side-chain. However, degradation can occur spontaneously (i.e. in the absence of enzymes). In this study, the influence of the first residue on the stability of a series of penultimate proline containing peptides, with the sequence Xaa-Pro-Gly-Gly (where Xaa is any amino acid), is investigated with mass spectrometry techniques. Peptides were incubated as mixtures at various solution temperatures (70℃ to 90℃) and were periodically sampled over the duration of the experiment. At elevated temperatures, we observe dissociation after the Xaa-Pro motif for all sequences, but at different rates. Transition state thermochemistry was obtained by studying the temperature-dependent kinetics and although all peptides show relatively small differences in the transition state free energies (∼95 kJ/mol), there is significant variability in the transition state entropy and enthalpy. This demonstrates that the side-chain of the first amino acid has a significant influence on the stability of the Xaa-Pro sequence. From these data, we demonstrate the ability to simultaneously measure the dissociation kinetics and relative transition state thermochemistries for a mixture of peptides, which vary only in the identity of the N-terminal amino acid.