Multiomics links global surfactant dysregulation with airflow obstruction and emphysema in COPD.

Rationale: Pulmonary surfactant is vital for lung homeostasis as it reduces surface tension to prevent alveolar collapse and provides essential immune-regulatory and antipathogenic functions. Previous studies demonstrated dysregulation of some individual surfactant components in COPD. We investigated relationships between COPD disease measures and dysregulation of surfactant components to gain new insights into potential disease mechanisms. Methods: Bronchoalveolar lavage proteome and lipidome were characterised in ex-smoking mild/moderate COPD subjects (n=26) and healthy ex-smoking (n=20) and never-smoking (n=16) controls using mass spectrometry. Serum surfactant protein analysis was performed. Results: Total phosphatidylcholine, phosphatidylglycerol, phosphatidylinositol, surfactant protein (SP)-B, SP-A and SP-D concentrations were lower in COPD versus controls (log2 fold change (log2FC) -2.0, -2.2, -1.5, -0.5, -0.7 and -0.5 (adjusted p<0.02), respectively) and correlated with lung function. Total phosphatidylcholine, phosphatidylglycerol, phosphatidylinositol, SP-A, SP-B, SP-D, napsin A and CD44 inversely correlated with computed tomography small airways disease measures (expiratory to inspiratory mean lung density) (r= -0.56, r= -0.58, r= -0.45, r= -0.36, r= -0.44, r= -0.37, r= -0.40 and r= -0.39 (adjusted p<0.05)). Total phosphatidylcholine, phosphatidylglycerol, phosphatidylinositol, SP-A, SP-B, SP-D and NAPSA inversely correlated with emphysema (% low-attenuation areas): r= -0.55, r= -0.61, r= -0.48, r= -0.51, r= -0.41, r= -0.31 and r= -0.34, respectively (adjusted p<0.05). Neutrophil elastase, known to degrade SP-A and SP-D, was elevated in COPD versus controls (log2FC 0.40, adjusted p=0.0390), and inversely correlated with SP-A and SP-D. Serum SP-D was increased in COPD versus healthy ex-smoking volunteers, and predicted COPD status (area under the curve 0.85). Conclusions: Using a multiomics approach, we demonstrate, for the first time, global surfactant dysregulation in COPD that was associated with emphysema, giving new insights into potential mechanisms underlying the cause or consequence of disease.

Sex-specific effects of CD248 on metabolism and the adipose tissue lipidome.

Cd248 has recently been associated with adipose tissue physiology, demonstrated by reduced weight gain in high fat diet-fed mice with genetic deletion of Cd248 relative to controls. Here we set out to determine the metabolic consequences of loss of Cd248. Strikingly, we find these to be sex specific; By subjecting Cd248-/- and Cd248+/+ mice to a high fat diet and indirect calorimetry study, we identified that only male Cd248-/- mice show reduced weight gain compared to littermate control wildtype mice. In addition, male (but not female) mice showed a lower respiratory exchange ratio on both chow and high fat diets, indicating a predisposition to metabolise lipid. Lipidomic studies on specific fat depots found reduced triglyceride and diglyceride deposition in male Cd248-/- mice, and this was supported by reduced expression of lipogenic and adipogenic genes. Finally, metabolomic analysis of isolated, differentiated preadipocytes found alterations in metabolic pathways associated with lipid deposition in cells isolated from male, but not female, Cd248-/- mice. Overall, our results highlight the importance of sex controls in animal studies and point to a role for Cd248 in sex- and depot-specific regulation of lipid metabolism.

Elevated Adipocyte Membrane Phospholipid Saturation Does Not Compromise Insulin Signaling.

Increased saturated fatty acid (SFA) levels in membrane phospholipids have been implicated in the development of metabolic disease. Here, we tested the hypothesis that increased SFA content in cell membranes negatively impacts adipocyte insulin signaling. Preadipocyte cell models with elevated SFA levels in phospholipids were generated by disrupting the ADIPOR2 locus, which resulted in a striking twofold increase in SFA-containing phosphatidylcholines and phosphatidylethanolamines, which persisted in differentiated adipocytes. Similar changes in phospholipid composition were observed in white adipose tissues isolated from the ADIPOR2-knockout mice. The SFA levels in phospholipids could be further increased by treating ADIPOR2-deficient cells with palmitic acid and resulted in reduced membrane fluidity and endoplasmic reticulum stress in mouse and human preadipocytes. Strikingly, increased SFA levels in differentiated adipocyte phospholipids had no effect on adipocyte gene expression or insulin signaling in vitro. Similarly, increased adipocyte phospholipid saturation did not impair white adipose tissue function in vivo, even in mice fed a high-saturated fat diet at thermoneutrality. We conclude that increasing SFA levels in adipocyte phospholipids is well tolerated and does not affect adipocyte insulin signaling in vitro and in vivo.

TLCD1 and TLCD2 regulate cellular phosphatidylethanolamine composition and promote the progression of non-alcoholic steatohepatitis.

The fatty acid composition of phosphatidylethanolamine (PE) determines cellular metabolism, oxidative stress, and inflammation. However, our understanding of how cells regulate PE composition is limited. Here, we identify a genetic locus on mouse chromosome 11, containing two poorly characterized genes Tlcd1 and Tlcd2, that strongly influences PE composition. We generated Tlcd1/2 double-knockout (DKO) mice and found that they have reduced levels of hepatic monounsaturated fatty acid (MUFA)-containing PE species. Mechanistically, TLCD1/2 proteins act cell intrinsically to promote the incorporation of MUFAs into PEs. Furthermore, TLCD1/2 interact with the mitochondria in an evolutionarily conserved manner and regulate mitochondrial PE composition. Lastly, we demonstrate the biological relevance of our findings in dietary models of metabolic disease, where Tlcd1/2 DKO mice display attenuated development of non-alcoholic steatohepatitis compared to controls. Overall, we identify TLCD1/2 proteins as key regulators of cellular PE composition, with our findings having broad implications in understanding and treating disease.

Engraftment of Bacteria after Fecal Microbiota Transplantation Is Dependent on Both Frequency of Dosing and Duration of Preparative Antibiotic Regimen.

The gut microbiota has emerged as a key mediator of human physiology, and germ-free mice have been essential in demonstrating a role for the microbiome in disease. Preclinical models using conventional mice offer the advantage of working with a mature immune system. However, optimal protocols for fecal microbiota transplant (FMT) engraftment in conventional mice are yet to be established. Conventional BALB/c mice were randomized to receive 3-day (3d) or 3-week (3w) antibiotic (ABX) regimen in their drinking water followed by 1 or 5-daily FMTs from a human donor. Fecal samples were collected longitudinally and characterized using 16S ribosomal RNA (rRNA) sequencing. Semi-targeted metabolomic profiling of fecal samples was also done with liquid chromatography-mass spectrometry (LC-MS). Lastly, we sought to confirm our findings in BKS mice. Recovery of baseline diversity scores were greatest in the 3d groups, driven by re-emergence of mouse commensal microbiota, whereas the most resemblance to donor microbiota was seen in the 3w + 5-FMT group. Amplicon sequence variants (ASVs) that were linked to the input material (human ASVs) engrafted to a significantly greater extent when compared to mouse ASVs in the 3-week groups but not the 3-day groups. Lastly, comparison of metabolomic profiles revealed distinct functional profiles by ABX regimen. These results indicate successful model optimization and emphasize the importance of ABX duration and frequency of FMT dosing; the most stable and reliable colonization by donor ASVs was seen in the 3wk + 5-FMT group.

Antibacterial Monoclonal Antibodies Do Not Disrupt the Intestinal Microbiome or Its Function.

Antibiotics revolutionized the treatment of infectious diseases; however, it is now clear that broad-spectrum antibiotics alter the composition and function of the host's microbiome. The microbiome plays a key role in human health, and its perturbation is increasingly recognized as contributing to many human diseases. Widespread broad-spectrum antibiotic use has also resulted in the emergence of multidrug-resistant pathogens, spurring the development of pathogen-specific strategies such as monoclonal antibodies (MAbs) to combat bacterial infection. Not only are pathogen-specific approaches not expected to induce resistance in nontargeted bacteria, but they are hypothesized to have minimal impact on the gut microbiome. Here, we compare the effects of antibiotics, pathogen-specific MAbs, and their controls (saline or control IgG [c-IgG]) on the gut microbiome of 7-week-old, female, C57BL/6 mice. The magnitude of change in taxonomic abundance, bacterial diversity, and bacterial metabolites, including short-chain fatty acids (SCFA) and bile acids in the fecal pellets from mice treated with pathogen-specific MAbs, was no different from that with animals treated with saline or an IgG control. Conversely, dramatic changes were observed in the relative abundance, as well as alpha and beta diversity, of the fecal microbiome and bacterial metabolites in the feces of all antibiotic-treated mice. Taken together, these results indicate that pathogen-specific MAbs do not alter the fecal microbiome like broad-spectrum antibiotics and may represent a safer, more-targeted approach to antibacterial therapy.

Recent advances in ion mobility-mass spectrometry for improved structural characterization of glycans and glycoconjugates.

Glycans and glycoconjugates are involved in regulating a vast array of cellular and molecular processes. Despite the importance of glycans in biology and disease, characterization of glycans remains difficult due to their structural complexity and diversity. Mass spectrometry (MS)-based techniques have emerged as the premier analytical tools for characterizing glycans. However, traditional MS-based strategies struggle to distinguish the large number of coexisting isomeric glycans that are indistinguishable by mass alone. Because of this, ion mobility spectrometry coupled to MS (IM-MS) has received considerable attention as an analytical tool for improving glycan characterization due to the capability of IM to resolve isomeric glycans before MS measurements. In this review, we present recent improvements in IM-MS instrumentation and methods for the structural characterization of isomeric glycans. In addition, we highlight recent applications of IM-MS that illustrate the enormous potential of this technology in a variety of research areas, including glycomics, glycoproteomics, and glycobiology.

Electron-Transfer/Higher-Energy Collision Dissociation (EThcD)-Enabled Intact Glycopeptide/Glycoproteome Characterization.

Protein glycosylation, one of the most heterogeneous post-translational modifications, can play a major role in cellular signal transduction and disease progression. Traditional mass spectrometry (MS)-based large-scale glycoprotein sequencing studies heavily rely on identifying enzymatically released glycans and their original peptide backbone separately, as there is no efficient fragmentation method to produce unbiased glycan and peptide product ions simultaneously in a single spectrum, and that can be conveniently applied to high throughput glycoproteome characterization, especially for N-glycopeptides, which can have much more branched glycan side chains than relatively less complex O-linked glycans. In this study, a redefined electron-transfer/higher-energy collision dissociation (EThcD) fragmentation scheme is applied to incorporate both glycan and peptide fragments in one single spectrum, enabling complete information to be gathered and great microheterogeneity details to be revealed. Fetuin was first utilized to prove the applicability with 19 glycopeptides and corresponding five glycosylation sites identified. Subsequent experiments tested its utility for human plasma N-glycoproteins. Large-scale studies explored N-glycoproteomics in rat carotid arteries over the course of restenosis progression to investigate the potential role of glycosylation. The integrated fragmentation scheme provides a powerful tool for the analysis of intact N-glycopeptides and N-glycoproteomics. We also anticipate this approach can be readily applied to large-scale O-glycoproteome characterization. Graphical Abstract ᅟ.

Targeted Mass Spectrometry Approach Enabled Discovery of O-Glycosylated Insulin and Related Signaling Peptides in Mouse and Human Pancreatic Islets.

O-Linked glycosylation often involves the covalent attachment of sugar moieties to the hydroxyl group of serine or threonine on proteins/peptides. Despite growing interest in glycoproteins, little attention has been directed to glycosylated signaling peptides, largely due to lack of enabling analytical tools. Here we explore the occurrence of naturally O-linked glycosylation on the signaling peptides extracted from mouse and human pancreatic islets using mass spectrometry (MS). A novel targeted MS-based method is developed to increase the likelihood of capturing these modified signaling peptides and to provide improved sequence coverage and accurate glycosite localization, enabling the first large-scale discovery of O-glycosylation on signaling peptides. Several glycosylated signaling peptides with multiple glycoforms are identified, including the first report of glycosylated insulin-B chain and insulin-C peptide and BigLEN. This discovery may reveal potential novel functions as glycosylation could influence their conformation and biostability. Given the importance of insulin and its related peptide hormones and previous studies of glycosylated insulin analogues, this natural glycosylation may provide important insights into diabetes research and therapeutic treatments.

A Database of Transition-Metal-Coordinated Peptide Cross-Sections: Selective Interaction with Specific Amino Acid Residues.

Ion mobility mass spectrometry (IMS-MS) techniques were used to generate a database of 2288 collision cross sections of transition-metal-coordinated tryptic peptide ions. This database consists of cross sections for 1253 [Pep + X]2+ and 1035 [Pep + X + H]3+, where X2+ corresponds to Mn2+, Co2+, Ni2+, Cu2+, or Zn2+. This number of measurements enables the extraction of structural trends for transition-metal-coordinated peptide ions. The range of structures and changes in collision cross sections for X2+-coordinated species (compared with protonated species of the same charge state) is similar to Mg2+-coordinated species. This suggests that the structures are largely determined by similarities in cation size with differences among the cross section distributions presumably caused by X2+ interactions with specific functional groups offered by the residue R-groups or the peptide backbone. Cross section contributions for individual residues upon X2+ solvation are assessed with the derivation of intrinsic size parameters (ISPs). The comparison of the [Pep + X]2+ ISPs with those previously reported for [Pep + Mg]2+ ions displays a lower contribution to the cross section for His, carboxyamidomethylated Cys, and Met, and is consistent with specific metal-residue interactions identified within protein X-ray crystallography databases. Graphical Abstract ᅟ.

Cis→Trans Isomerization of Pro(7) in Oxytocin Regulates Zn(2+) Binding.

Ion mobility/mass spectrometry techniques are employed to investigate the binding of Zn(2+) to the nine-residue peptide hormone oxytocin (OT, Cys(1)-Tyr(2)-Ile(3)-Gln(4)-Asn(5)-Cys(6)-Pro(7)-Leu(8)-Gly(9)-NH2, having a disulfide bond between Cys(1) and Cys(6) residues). Zn(2+) binding to OT is known to increase the affinity of OT for its receptor [Pearlmutter, A. F., Soloff, M. S.: Characterization of the metal ion requirement for oxytocin-receptor interaction in rat mammary gland membranes. J. Biol. Chem. 254, 3899-3906 (1979)]. In the absence of Zn(2+), we find evidence for two primary OT conformations, which arise because the Cys(6)-Pro(7) peptide bond exists in both the trans- and cis-configurations. Upon addition of Zn(2+), we determine binding constants in water of KA = 1.43 ± 0.24 and 0.42 ± 0.12 µM(-1), for the trans- and cis-configured populations, respectively. The Zn(2+) bound form of OT, having a cross section of Ω = 235 Å(2), has Pro(7) in the trans-configuration, which agrees with a prior report [Wyttenbach, T., Liu, D., Bowers, M. T.: Interactions of the hormone oxytocin with divalent metal ions. J. Am. Chem. Soc. 130, 5993-6000 (2008)], in which it was proposed that Zn(2+) binds to the peptide ring and is further coordinated by interaction of the C-terminal, Pro(7)-Leu(8)-Gly(9)-NH2, tail. The present work shows that the cis-configuration of OT isomerizes to the trans-configuration upon binding Zn(2+). In this way, the proline residue regulates Zn(2+) binding to OT and, hence, is important in receptor binding. Graphical Abstract ᅟ.

Examining the Influence of Phosphorylation on Peptide Ion Structure by Ion Mobility Spectrometry-Mass Spectrometry.

Ion mobility spectrometry-mass spectrometry (IMS-MS) techniques are used to study the general effects of phosphorylation on peptide structure. Cross sections for a library of 66 singly phosphorylated peptide ions from 33 pairs of positional isomers, and unmodified analogues were measured. Intrinsic size parameters (ISPs) derived from these measurements yield calculated collision cross sections for 85% of these phosphopeptide sequences that are within ±2.5% of experimental values. The average ISP for the phosphoryl group (0.64 ± 0.05) suggests that in general this moiety forms intramolecular interactions with the neighboring residues and peptide backbone, resulting in relatively compact structures. We assess the capability of ion mobility to separate positional isomers (i.e., peptide sequences that differ only in the location of the modification) and find that more than half of the isomeric pairs have >1% difference in collision cross section. Phosphorylation is also found to influence populations of structures that differ in the cis/trans orientation of Xaa-Pro peptide bonds. Several sequences with phosphorylated Ser or Thr residues located N-terminally adjacent to Pro residues show fewer conformations compared to the unmodified sequences.

Hybrid ion mobility and mass spectrometry as a separation tool.

Ion mobility spectrometry (IMS) coupled to mass spectrometry (MS) has seen spectacular growth over the last two decades. Increasing IMS sensitivity and capacity with improvements in MS instrumentation have driven this growth. As a result, a diverse new set of techniques for separating ions by their mobility have arisen, each with characteristics that make them favorable for some experiments and some mass spectrometers. Ion mobility techniques can be broken down into dispersive and selective techniques based upon whether they pass through all mobilities for later analysis by mass spectrometry or select ions by mobility or a related characteristic. How ion mobility techniques fit within a more complicated separation including mass spectrometry and other techniques such as liquid chromatography is of fundamental interest to separations scientists. In this review we explore the multitude of ion mobility techniques hybridized to different mass spectrometers, detailing current challenges and opportunities for each ion mobility technique and for what experiments one technique might be chosen over another. The underlying principles of ion mobility separations, including: considerations regarding separation capabilities, ion transmission, signal intensity and sensitivity, and the impact that the separation has upon the ion structure (i.e., the possibility of configurational changes due to ion heating) are discussed.

Ion Mobility-Mass Spectrometry Reveals the Energetics of Intermediates that Guide Polyproline Folding.

Proline favors trans-configured peptide bonds in native proteins. Although cis/trans configurations vary for non-native and unstructured states, solvent also influences these preferences. Water induces the all-cis right-handed polyproline-I (PPI) helix of polyproline to fold into the all-trans left-handed polyproline-II (PPII) helix. Our recent work has shown that this occurs via a sequential mechanism involving six resolved intermediates [Shi, L., Holliday, A.E., Shi, H., Zhu, F., Ewing, M.A., Russell, D.H., Clemmer, D.E.: Characterizing intermediates along the transition from PPI to PPII using ion mobility-mass spectrometry. J. Am. Chem. Soc. 136, 12702-12711 (2014)]. Here, we use ion mobility-mass spectrometry to make the first detailed thermodynamic measurements of the folding intermediates, which inform us about how and why this transition occurs. It appears that early intermediates are energetically favorable because of the hydration of the peptide backbone, whereas late intermediates are enthalpically unfavorable. However, folding continues, as the entropy of the system increases upon successive formation of each new structure. When PPII is immersed in 1-propanol, the PPII→PPI transition occurs, but this reaction occurs through a very different mechanism. Early on, the PPII population splits onto multiple pathways that eventually converge through a late intermediate that continues on to the folded PPI helix. Nearly every step is endothermic. Folding results from a stepwise increase in the disorder of the system, allowing a wide-scale search for a critical late intermediate. Overall, the data presented here allow us to establish the first experimentally determined energy surface for biopolymer folding as a function of solution environment.

Photosynthesis of a combinatorial peptide library in the gas phase.

A strategy for generating large numbers of peptides from a relatively small number of precursors based on photosynthetic combination in the gas phase is presented. In this approach, electrospray ionization is used to create a combination of proton-bound dimers from a specified set of peptides present in solution. The dimers are then accumulated and isolated in an ion trap mass spectrometer. Photoexcitation (at 157 nm) leads to water elimination and the formation of larger peptide sequences that are characterized by subsequent isolation and collision-induced dissociation. The method is illustrated by using a set of four enkephalin-related and acetylated peptides to generate 12 larger peptide sequences. The ability to synthesize, isolate, and characterize many amino acid sequences from only a few precursors provides a fast and efficient means of characterizing properties of such species (e.g., dissociation patterns and reactivities).

Penultimate proline in neuropeptides.

A recent ion mobility spectrometry-mass spectrometry (IMS-MS) study revealed that tryptic peptide ions containing a proline residue at the second position from the N-terminus (i.e., penultimate proline) frequently adopt multiple conformations, owing to the cis-trans isomerization of Xaa(1)-Pro(2) peptide bonds [J. Am. Soc. Mass Spectrom. 2015, 26, 444]. Here, we present a statistical analysis of a neuropeptide database that illustrates penultimate proline residues are frequently found in neuropeptides. In order to probe the effect of penultimate proline on neuropeptide conformations, IMS-MS experiments were performed on two model peptides in which penultimate proline residues were known to be important for biological activity: the N-terminal region of human neuropeptide Y (NPY1-9, Tyr(1)-Pro(2)-Ser(3)-Lys(4)-Pro(5)-Asp(6)-Asn(7)-Pro(8)-Gly(9)-NH2) and a tachykinin-related peptide (CabTRP Ia, Ala(1)-Pro(2)-Ser(3)-Gly(4)-Phe(5)-Leu(6)-Gly(7)-Met(8)-Arg(9)-NH2). From these studies, it appears that penultimate prolines allow neuropeptides to populate multiple conformations arising from the cis-trans isomerization of Xaa(1)-Pro(2) peptide bonds. Although it is commonly proposed that the role of penultimate proline residues is to protect peptides from enzymatic degradation, the present results indicate that penultimate proline residues also are an important means of increasing the conformational heterogeneity of neuropeptides.

Infrared Spectroscopy of Mobility-Selected H+-Gly-Pro-Gly-Gly (GPGG).

We report the first results from a new instrument capable of acquiring infrared spectra of mobility-selected ions. This demonstration involves using ion mobility to first separate the protonated peptide Gly-Pro-Gly-Gly (GPGG) into two conformational families with collisional cross-sections of 93.8 and 96.8 Å(2). After separation, each family is independently analyzed by acquiring the infrared predissociation spectrum of the H(2)-tagged molecules. The ion mobility and spectroscopic data combined with density functional theory (DFT) based molecular dynamics simulations confirm the presence of one major conformer per family, which arises from cis/trans isomerization about the proline residue. We induce isomerization between the two conformers by using collisional activation in the drift tube and monitor the evolution of the ion distribution with ion mobility and infrared spectroscopy. While the cis-proline species is the preferred gas-phase structure, its relative population is smaller than that of the trans-proline species in the initial ion mobility drift distribution. This suggests that a portion of the trans-proline ion population is kinetically trapped as a higher energy conformer and may retain structural elements from solution.

Conformational landscape and pathway of disulfide bond reduction of human alpha defensin.

Human alpha defensins are a class of antimicrobial peptides with additional antiviral activity. Such antimicrobial peptides constitute a major part of mammalian innate immunity. Alpha defensins contain six cysteines, which form three well defined disulfide bridges under oxidizing conditions. Residues C3-C31, C5-C20, and C10-C30 form disulfide pairs in the native structure of the peptide. The major tissue in which HD5 is expressed is the crypt of the small intestine, an anaerobic niche that should allow for substantial pools of both oxidized and (partly) reduced HD5. We used ion mobility coupled to mass spectrometry to track the structural changes in HD5 upon disulfide bond reduction. We found evidence of stepwise unfolding of HD5 with sequential reduction of the three disulfide bonds. Alkylation of free cysteines followed by tandem mass spectrometry of the corresponding partially reduced states revealed a dominant pathway of reductive unfolding. The majority of HD5 unfolds by initial reduction of C5-C20, followed by C10-C30 and C3-C31. We find additional evidence for a minor pathway that starts with reduction of C3-C31, followed by C5-C20 and C10-C30. Our results provide insight into the pathway and conformational landscape of disulfide bond reduction in HD5.

Populations of metal-glycan structures influence MS fragmentation patterns.

The structures and collision-induced dissociation (CID) fragmentation patterns of the permethylated glycan Man5GlcNAc2 are investigated by a combination of hybrid ion mobility spectrometry (IMS), mass spectrometry (MS), and MS/MS techniques. IMS analysis of eight metal-adducted glycans ([Man5GlcNAc2 + M](2+), where M = Mn, Fe, Co, Ni, Cu, Mg, Ca, and Ba) shows distinct conformer patterns. These conformers appear to arise from individual metals binding at different sites on the glycan. Fragmentation studies suggest that these different binding sites influence the CID fragmentation patterns. This paper describes a series of separation, activation, and fragmentation studies that assess which fragments arise from each of the different gas-phase conformer states. Comparison of the glycan distributions formed under gentle ionization conditions with those obtained after activation of the gas-phase ions suggests that these conformer binding states also appear to exist in solution.

On the split personality of penultimate proline.

The influence of the position of the amino acid proline in polypeptide sequences is examined by a combination of ion mobility spectrometry-mass spectrometry (IMS-MS), amino acid substitutions, and molecular modeling. The results suggest that when proline exists as the second residue from the N-terminus (i.e., penultimate proline), two families of conformers are formed. We demonstrate the existence of these families by a study of a series of truncated and mutated peptides derived from the 11-residue peptide Ser(1)-Pro(2)-Glu(3)-Leu(4)-Pro(5)-Ser(6)-Pro(7)-Gln(8)-Ala(9)-Glu(10)-Lys(11). We find that every peptide from this sequence with a penultimate proline residue has multiple conformations. Substitution of Ala for Pro residues indicates that multiple conformers arise from the cis-trans isomerization of Xaa(1)-Pro(2) peptide bonds as Xaa-Ala peptide bonds are unlikely to adopt the cis isomer, and examination of spectra from a library of 58 peptides indicates that ~80% of sequences show this effect. A simple mechanism suggesting that the barrier between the cis- and trans-proline forms is lowered because of low steric impedance is proposed. This observation may have interesting biological implications as well, and we note that a number of biologically active peptides have penultimate proline residues.