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RATIONALE: Hydrogen/deuterium exchange mass spectrometry (HDX-MS) can provide precise analysis of a protein's conformational dynamics across varied states, such as heat-denatured versus native protein structures, localizing regions that are specifically affected by such conditional changes. Maximizing protein sequence coverage provides high confidence that regions of interest were located by HDX-MS, but one challenge for complete sequence coverage is N-glycosylation sites. The deuteration of peptides post-translationally modified by asparagine-bound glycans (glycopeptides) has not always been identified in previous reports of HDX-MS analyses, causing significant sequence coverage gaps in heavily glycosylated proteins and uncertainty in structural dynamics in many regions throughout a glycoprotein. METHODS: We detected deuterated glycopeptides with a Tribrid Orbitrap Eclipse mass spectrometer performing data-dependent acquisition. An MS scan was used to identify precursor ions; if high-energy collision-induced dissociation MS/MS of the precursor indicated oxonium ions diagnostic for complex glycans, then electron transfer low-energy collision-induced dissociation MS/MS scans of the precursor identified the modified asparagine residue and the glycan's mass. As in traditional HDX-MS, the identified glycopeptides were then analyzed at the MS level in samples labeled with D2 O. RESULTS: We report HDX-MS analysis of the SARS-CoV-2 spike protein ectodomain in its trimeric prefusion form, which has 22 predicted N-glycosylation sites per monomer, with and without heat treatment. We identified glycopeptides and calculated their average isotopic mass shifts from deuteration. Inclusion of the deuterated glycopeptides increased sequence coverage of spike ectodomain from 76% to 84%, demonstrated that glycopeptides had been deuterated, and improved confidence in results localizing structural rearrangements. CONCLUSION: Inclusion of deuterated glycopeptides improves the analysis of the conformational dynamics of glycoproteins such as viral surface antigens and cellular receptors.
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COVID-19 , Glicopéptidos , Humanos , Glicopéptidos/química , Glicoproteína de la Espiga del Coronavirus , Espectrometría de Masas en Tándem/métodos , Deuterio , SARS-CoV-2 , Asparagina , Glicoproteínas/química , Polisacáridos , IonesRESUMEN
N-glycosylation plays an important role in the structure and function of membrane and secreted proteins. The spike protein on the surface of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes COVID-19, is heavily glycosylated and the major target for developing vaccines, therapeutic drugs and diagnostic tests. The first major SARS-CoV-2 variant carries a D614G substitution in the spike (S-D614G) that has been associated with altered conformation, enhanced ACE2 binding, and increased infectivity and transmission. In this report, we used mass spectrometry techniques to characterize and compare the N-glycosylation of the wild type (S-614D) or variant (S-614G) SARS-CoV-2 spike glycoproteins prepared under identical conditions. The data showed that half of the N-glycosylation sequons changed their distribution of glycans in the S-614G variant. The S-614G variant showed a decrease in the relative abundance of complex-type glycans (up to 45%) and an increase in oligomannose glycans (up to 33%) on all altered sequons. These changes led to a reduction in the overall complexity of the total N-glycosylation profile. All the glycosylation sites with altered patterns were in the spike head while the glycosylation of three sites in the stalk remained unchanged between S-614G and S-614D proteins.
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Glicopéptidos/análisis , Espectrometría de Masas/métodos , SARS-CoV-2/metabolismo , Glicoproteína de la Espiga del Coronavirus/metabolismo , Enzima Convertidora de Angiotensina 2/química , Enzima Convertidora de Angiotensina 2/metabolismo , COVID-19/patología , COVID-19/virología , Cromatografía Líquida de Alta Presión , Glicosilación , Humanos , Mutación , Unión Proteica , Estructura Terciaria de Proteína , SARS-CoV-2/aislamiento & purificación , Glicoproteína de la Espiga del Coronavirus/químicaRESUMEN
Genomic sequence and gene expression association studies in animals and humans have identified genes that may be integral in the pathogenesis of various diseases. CD14 (cluster of differentiation 14)-a cell surface protein involved in innate immune system activation-is one such gene associated with cardiovascular and hypertensive disease. We previously showed that this gene is upregulated in renal macrophages of Dahl salt-sensitive animals fed a high-salt diet; here we test the hypothesis that CD14 contributes to the elevated pressure and renal injury observed in salt-sensitive hypertension. Using CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat-associated 9), we created a targeted mutation in the CD14 gene on the Dahl SS (SS/JrHSDMcwi) background and validated the absence of CD14 peptides via mass spectrometry. Radiotelemetry was used to monitor blood pressure in wild-type and CD14-/- animals challenged with high salt and identified infiltrating renal immune cells via flow cytometry. Germline knockout of CD14 exacerbated salt-sensitive hypertension and renal injury in female animals but not males. CD14-/- females demonstrated increased infiltrating macrophages but no difference in infiltrating lymphocytes. Transplant of CD14+/+ or CD14-/- bone marrow was used to isolate the effects of CD14 knockout to hematopoietic cells and confirmed that the differential phenotype observed was due to knockout of CD14 in hematopoietic cells. Ovariectomy was used to remove the influence of female sex hormones, which completely abrogated the effect of CD14 knockout. These studies provide a novel treatment target and evidence of a new dichotomy in immune activation between sexes within the context of hypertensive disease where CD14 regulates immune cell activation and renal injury.
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Hipertensión/inmunología , Riñón/patología , Receptores de Lipopolisacáridos/fisiología , Caracteres Sexuales , Lesión Renal Aguda , Animales , Estradiol/fisiología , Femenino , Hipertensión/complicaciones , Receptores de Lipopolisacáridos/genética , Masculino , Ratas , Ratas Endogámicas DahlRESUMEN
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to a global pandemic of coronavirus disease 2019 (COVID-19). The spike protein expressed on the surface of this virus is highly glycosylated and plays an essential role during the process of infection. We conducted a comprehensive mass spectrometric analysis of the N-glycosylation profiles of the SARS-CoV-2 spike proteins using signature ions-triggered electron-transfer/higher-energy collision dissociation (EThcD) mass spectrometry. The patterns of N-glycosylation within the recombinant ectodomain and S1 subunit of the SARS-CoV-2 spike protein were characterized using this approach. Significant variations were observed in the distribution of glycan types as well as the specific individual glycans on the modification sites of the ectodomain and subunit proteins. The relative abundance of sialylated glycans in the S1 subunit compared to the full-length protein could indicate differences in the global structure and function of these two species. In addition, we compared N-glycan profiles of the recombinant spike proteins produced from different expression systems, including human embryonic kidney (HEK 293) cells and Spodoptera frugiperda (SF9) insect cells. These results provide useful information for the study of the interactions of SARS-CoV-2 viral proteins and for the development of effective vaccines and therapeutics.
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Betacoronavirus/química , Polisacáridos/análisis , Glicoproteína de la Espiga del Coronavirus/química , Animales , Glicosilación , Células HEK293 , Humanos , Espectrometría de Masas/métodos , Polisacáridos/química , SARS-CoV-2 , Spodoptera/químicaRESUMEN
BACKGROUND: Multiple myeloma (MM) is characterized by clonal expansion of malignant plasma cells in the bone marrow. While recent advances in treatment for MM have improved patient outcomes, the 5-year survival rate remains ~50%. A better understanding of the MM cell surface proteome could facilitate development of new directed therapies and assist in stratification and monitoring of patient outcomes. METHODS: In this study, we first used a mass spectrometry (MS)-based discovery-driven cell surface capture (CSC) approach to map the cell surface N-glycoproteome of MM cell lines. Next, we developed targeted MS assays, and applied these to cell lines and primary patient samples to refine the list of candidate tumor markers. Candidates of interest detected by MS on MM patient samples were further validated using flow cytometry (FCM). RESULTS: We identified 696 MM cell surface N-glycoproteins by CSC, and developed 73 targeted MS detection assays. MS-based validation using primary specimens detected 30 proteins with significantly higher abundance in patient MM cells than controls. Nine of these proteins were identified as potential immunotherapeutic targets, including five that were validated by FCM, confirming their expression on the cell surface of primary MM patient cells. CONCLUSIONS: This MM surface N-glycoproteome will be a valuable resource in the development of biomarkers and therapeutics. Further, we anticipate that our targeted MS assays will have clinical benefit for the diagnosis, stratification, and treatment of MM patients.
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Biomarcadores de Tumor/sangre , Inmunoterapia/métodos , Glicoproteínas de Membrana/metabolismo , Línea Celular , Femenino , Humanos , MasculinoRESUMEN
The E3 ubiquitin ligase C-terminus of Hsc70 interacting protein (CHIP) plays a critical role in regulating the ubiquitin-dependent degradation of misfolded proteins. CHIP mediates the ubiquitination of the α-amino-terminus of substrates with the E2 Ube2w and facilitates the ubiquitination of lysine residues with the E2 UbcH5. While it is known that Ube2w directly interacts with the disordered regions at the N-terminus of its substrates, it is unclear how CHIP and UbcH5 mediate substrate lysine selection. Here, we have decoupled the contributions of the E2, UbcH5, and the E3, CHIP, in ubiquitin transfer. We show that UbcH5 selects substrate lysine residues independent of CHIP, and that CHIP participates in lysine selection by fine-tuning the subset of substrate lysines that are ubiquitinated. We also identify lysine 128 near the C-terminus of UbcH5 as a critical residue for the efficient ubiquitin transfer by UbcH5 in both the presence and absence of CHIP. Together, these data demonstrate an important role of the UbcH5/substrate interactions in mediating the efficient ubiquitin transfer by the CHIP/UbcH5 complex.
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Lisina/metabolismo , Enzimas Ubiquitina-Conjugadoras/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Humanos , Lisina/química , Modelos Moleculares , Enzimas Ubiquitina-Conjugadoras/química , Ubiquitina-Proteína Ligasas/química , UbiquitinaciónRESUMEN
Compared to adults, children experience increased postoperative scarring and inflammation following intraocular surgery. While the underlying causes of the exaggerated immune response in children are not understood, proteins play key roles in postoperative scarring and wound healing processes. To identify and quantify proteins associated with the robust postoperative immune response, this study applied quantitative proteomics approaches to a juvenile rabbit model of lensectomy with intraocular lens (IOL) insertion. Twenty-six 6-7 week-old New Zealand white rabbits underwent unilateral portions of lensectomy with IOL insertion including: anterior chamber paracentesis, corneal incision with wound suture, lensectomy only, and lensectomy with IOL insertion. Aqueous humor was collected immediately prior and three days after each procedure. Semi-quantitative protein discovery was achieved by label-free quantitation using data dependent and data independent acquisition modes. Based on the discovery results, targeted quantitation by parallel reaction monitoring of 3 proteins of interest, fibrinogen-beta chain, transforming growth factor beta-2, and retinol binding protein 3, was used to confirm the observed quantitative trends. Total protein concentration levels increased with each progressive surgical step of lensectomy with IOL insertion. Proteins related to the complement and coagulation cascades were found to increase in relative abundance, while proteins related to ocular immunosuppression decreased in abundance following surgery. These data provide insights into the postoperative response by providing the first surgical step-wise views of the AH proteome before and after surgery. Overall, this work provides the foundation for future investigations targeting specific proteins for therapeutic interventions aimed at minimizing postoperative complications after pediatric intraocular surgery.
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Humor Acuoso/metabolismo , Inflamación/metabolismo , Implantación de Lentes Intraoculares/efectos adversos , Cristalino/cirugía , Proteómica/métodos , Animales , Modelos Animales de Enfermedad , Proteínas del Ojo/metabolismo , Fibrinógeno/metabolismo , Inflamación/etiología , Masculino , Conejos , Proteínas de Unión al Retinol/metabolismo , Suturas/efectos adversos , Factor de Crecimiento Transformador beta2/metabolismo , Regulación hacia ArribaRESUMEN
Mechanotransduction refers to the processes whereby mechanical stimuli are converted into electrochemical signals that allow for the sensation of our surrounding environment through touch. Despite its fundamental role in our daily lives, the molecular and cellular mechanisms of mechanotransduction are not yet well-defined. Previous data suggest that keratinocytes may release factors that activate or modulate cutaneous sensory neuron terminals, including small molecules, lipids, peptides, proteins, and oligosaccharides. This study presents a first step toward identifying soluble mediators of keratinocyte-sensory neuron communication by evaluating the potential for top-down mass spectrometry to identify proteoforms released during 1 min of mechanical stimulation of mouse skin from naiÌve animals. Overall, this study identified 47 proteoforms in the secretome of mouse hind paw skin, of which 14 were differentially released during mechanical stimulation, and includes proteins with known and previously unknown relevance to mechanotransduction. Finally, this study outlines a bioinformatic workflow that merges output from two complementary analysis platforms for top-down data and demonstrates the utility of this workflow for integrating quantitative and qualitative data.
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Espectrometría de Masas/métodos , Mecanotransducción Celular , Proteínas/análisis , Piel/metabolismo , Animales , Biología Computacional , Queratinocitos/metabolismo , Ratones , Proteómica/métodos , Piel/química , Flujo de TrabajoRESUMEN
Lymphocytes are immune cells that are critical for the maintenance of adaptive immunity. Differentiation of lymphoid progenitors yields B-, T-, and NK-cell subtypes that individually correlate with specific forms of leukemia or lymphoma. Therefore, it is imperative a precise method of cell categorization is utilized to detect differences in distinct disease states present in patients. One viable means of classification involves evaluation of the cell surface proteome of lymphoid malignancies. Specifically, this manuscript details the use of an antibody independent approach known as Cell Surface Capture Technology, to assess the N-glycoproteome of four human lymphocyte cell lines. Altogether, 404 cell surface N-glycoproteins were identified as markers for specific cell types involved in lymphocytic malignancies, including 82 N-glycoproteins that had not been previously been described for B or T cells within the Cell Surface Protein Atlas. Comparative analysis, hierarchical clustering techniques, and label-free quantitation were used to reveal proteins most informative for each cell type. Undoubtedly, the characterization of the cell surface proteome of lymphoid malignancies is a first step toward improving personalized diagnosis and treatment of leukemia and lymphoma.
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Biomarcadores de Tumor/metabolismo , Membrana Celular/metabolismo , Glicoproteínas/metabolismo , Leucemia/metabolismo , Linfocitos/metabolismo , Linfoma/metabolismo , Proteoma/análisis , Células Cultivadas , Humanos , Leucemia/patología , Linfocitos/citología , Linfoma/patología , Proteómica/métodosRESUMEN
The epidermal growth factor receptor (EGFR)/ErbB family of receptor tyrosine kinases includes oncogenes important in the progression of breast and other cancers, and they are targets for many drug development strategies. Each member of the ErbB family possesses a unique, structurally uncharacterized C-terminal tail that plays an important role in autophosphorylation and signal propagation. To determine whether these C-terminal tails are intrinsically disordered regions, we conducted a battery of biophysical experiments on the EGFR and HER3 tails. Using hydrogen/deuterium exchange mass spectrometry, we measured the conformational dynamics of intracellular half constructs and compared the tails with the ordered kinase domains. The C-terminal tails demonstrate more rapid deuterium exchange behavior when compared with the kinase domains. Next, we expressed and purified EGFR and HER3 tail-only constructs. Results from circular dichroism spectroscopy, size exclusion chromatography with multiangle light scattering, dynamic light scattering, analytical ultracentrifugation, and small angle X-ray scattering each provide evidence that the EGFR and HER3 C-terminal tails are intrinsically disordered with extended, non-globular structure in solution. The intrinsic disorder and extended conformation of these tails may be important for their function by increasing the capture radius and reducing the thermodynamic barriers for binding of downstream signaling proteins.
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Receptores ErbB/química , Receptor ErbB-3/química , Dicroismo Circular , Medición de Intercambio de Deuterio , Receptores ErbB/genética , Receptores ErbB/metabolismo , Humanos , Cinética , Dominios Proteicos , Receptor ErbB-3/genética , Receptor ErbB-3/metabolismo , Difracción de Rayos XRESUMEN
Measurement of residual structure in intrinsically disordered proteins can provide insights into the mechanisms by which such proteins undergo coupled binding and folding. The present work describes an approach to measure residual structure in disordered proteins using millisecond hydrogen/deuterium (H/D) exchange in a conventional bottom-up peptide-based workflow. We used the exchange mid-point, relative to a totally deuterated control, to quantify the rate of H/D exchange in each peptide. A weighted residue-by-residue average of these midpoints was used to map the extent of residual structure at near single-residue resolution. We validated this approach both by simulating a disordered protein and experimentally using the p300 binding domain of ACTR, a model disordered protein already well-characterized by other approaches. Secondary structure elements mapped in the present work are in good agreement with prior nuclear magnetic resonance measurements. The new approach was somewhat limited by a loss of spatial resolution and subject to artifacts because of heterogeneities in intrinsic exchange. Approaches to correct these limitations are discussed.
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Medición de Intercambio de Deuterio/métodos , Proteínas Intrínsecamente Desordenadas/análisis , Proteínas Intrínsecamente Desordenadas/química , Espectrometría de Masas/métodos , Mapeo Peptídico/métodos , Estructura Secundaria de ProteínaRESUMEN
The ability to selectively activate function of particular proteins via pharmacological agents is a longstanding goal in chemical biology. Recently, we reported an approach for designing a de novo allosteric effector site directly into the catalytic domain of an enzyme. This approach is distinct from traditional chemical rescue of enzymes in that it relies on disruption and restoration of structure, rather than active site chemistry, as a means to achieve modulate function. However, rationally identifying analogous de novo binding sites in other enzymes represents a key challenge for extending this approach to introduce allosteric control into other enzymes. Here we show that mutation sites leading to protein inactivation via tryptophan-to-glycine substitution and allowing (partial) reactivation by the subsequent addition of indole are remarkably frequent. Through a suite of methods including a cell-based reporter assay, computational structure prediction and energetic analysis, fluorescence studies, enzymology, pulse proteolysis, X-ray crystallography, and hydrogen-deuterium mass spectrometry, we find that these switchable proteins are most commonly modulated indirectly, through control of protein stability. Addition of indole in these cases rescues activity not by reverting a discrete conformational change, as we had observed in the sole previously reported example, but rather rescues activity by restoring protein stability. This important finding will dramatically impact the design of future switches and sensors built by this approach, since evaluating stability differences associated with cavity-forming mutations is a far more tractable task than predicting allosteric conformational changes. By analogy to natural signaling systems, the insights from this study further raise the exciting prospect of modulating stability to design optimal recognition properties into future de novo switches and sensors built through chemical rescue of structure.
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Proteínas/química , Electroforesis en Gel de Poliacrilamida , Genes Reporteros , Proteínas Fluorescentes Verdes/química , Indoles/química , Mutación , Conformación ProteicaRESUMEN
Measurement of amide H/D exchange on the ms time scale can provide valuable information about the dynamic behavior of the most flexible regions of proteins. We describe here a simple mixing apparatus, assembled solely from off-the-shelf components, that can be used for H/D exchange mass spectrometry to measure exchange on the 50-5000 ms time scale. Our apparatus utilizes flow-injection to minimize sample consumption. Although the mixer operates at low Reynolds numbers (less than 10(2)) where laminar flow is expected, H/D exchange kinetics were well-approximated using the assumption of plug-flow. We validated this approximation using fluorescence imaging of fluorescein-conjugated bovine serum albumin in the delay line and by demonstrating agreement between measured and calculated H/D exchange kinetics for a mixture of peptides. The performance of the apparatus was further validated by measuring rapid H/D exchange kinetics by an intrinsically disordered protein, murine CBP(2059-2117) (UniProt CBP_MOUSE). H/D exchange data from CBP, both free and in complex with human ACTR(1018-1088) (UniProt NCOA3_HUMAN), were consistent with previous biophysical studies of this protein.
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Proteína de Unión a CREB/química , Medición de Intercambio de Deuterio/instrumentación , Secuencia de Aminoácidos , Animales , Humanos , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Factores de TiempoRESUMEN
Mapping the structured and disordered regions and identifying disorder-to-order transitions are essential to understanding intrinsically disordered proteins (IDPs). One technique that can provide such information is H/D exchange coupled with mass spectrometry (H/D-MS). To explore the feasibility of H/D-MS for mapping disordered and ordered regions in IDPs, we undertook a systematic evaluation of an unstructured protein, a molten globular protein, and the well-folded complex of the two proteins. Most segments of the unstructured protein, ACTR (activator of thyroid and retinoid receptors, NCOA3_HUMAN, residues 1018-1088), exchange at rates consistent with its assignment as an unstructured protein, but there is slight protection in regions that become helical in the ACTR-CBP complex. The molten globular protein, CBP (the nuclear coactivator binding domain of the CREB binding protein, CBP_MOUSE, residues 2059-2117), is moderately protected from exchange, and the protection is nearly uniform across the length of the protein. The uniformity arises because of rapid interconversion between an ensemble of folded conformers and an ensemble of unstructured conformers. Rapid interconversion causes the H/D exchange kinetics to be dominated by exchange by molecules in unstructured conformations. For the folded ACTR-CBP complex, the exchange data provide a qualitatively accurate description of the complex. Our results provide a useful framework to use in the interpretation of H/D-MS data of intrinsically disordered proteins.
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Proteína de Unión a CREB/química , Espectrometría de Masas/métodos , Coactivador 3 de Receptor Nuclear/química , Animales , Proteína de Unión a CREB/genética , Proteína de Unión a CREB/metabolismo , Deuterio/química , Humanos , Hidrógeno/química , Marcaje Isotópico , Modelos Moleculares , Coactivador 3 de Receptor Nuclear/genética , Coactivador 3 de Receptor Nuclear/metabolismo , Pliegue de ProteínaRESUMEN
Loss of deuterium label during the LC step in amide hydrogen/deuterium exchange mass spectrometry (H/D-MS) is minimized by maintaining an acidic mobile phase pH and low temperature (pH 2.5, 0 °C). Here we detail the construction and performance of a low-cost, thermoelectrically refrigerated enclosure to house high-performance liquid chromatography (HPLC) components and cool mobile phases. Small volume heat exchangers rapidly decrease mobile phase temperature and keep the temperature stable to ±0.2 °C. Using a superficially porous reversed-phase column, we obtained excellent chromatographic performance in the separation of peptides with a median peak width of 4.4 s. Average deuterium recovery was 80.2% with an average relative precision of 0.91%.
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Cromatografía de Fase Inversa/métodos , Medición de Intercambio de Deuterio/métodos , Espectrometría de Masas/métodos , Refrigeración/instrumentación , Secuencia de Aminoácidos , Cromatografía Líquida de Alta Presión , Cromatografía de Fase Inversa/instrumentación , Diseño de Equipo , Concentración de Iones de Hidrógeno , Datos de Secuencia Molecular , Péptidos/química , TemperaturaRESUMEN
The recent worldwide shortage of acetonitrile has prompted interest in alternative solvents for liquid chromatography/mass spectrometry (LC/MS). In this work, acetone was substituted for acetonitrile in the separation of a peptide mixture by reversed-phase high-performance liquid chromatography (RP-HPLC) and in the positive electrospray ionization mass spectrometry (ESI-MS) of individual peptides. On both C12 and C18 stationary phases, the substitution of acetone for acetonitrile as the organic component of the mobile phase did not alter the gradient elution order of a five-peptide retention standard, but did increase peak width, shorten retention times, and increase peak tailing. Positive ESI mass spectra were obtained for angiotensin I, bradykinin, [Leu(5)]-enkephalin, and somatostatin 14 dissolved in both acetonitrile/water/formic acid (25%/75%/0.1%) and acetone/water/formic acid (25%/75%/0.1%). Under optimized ESI-MS conditions, the mass spectral response of [Leu(5)]-enkephalin was increased two-fold when the solvent contained acetone. The substitution of acetone for acetonitrile resulted in only slight changes in the responses of the remaining peptides. A higher capillary voltage was required for optimum response when acetone was used. Compared with acetonitrile/water/formic acid (50/50/0.1%), more interfering species below m/z = 140 were found in the ESI-MS spectra of acetone/water/formic acid (50/50/0.1%).