Priority Outcomes in Sickle Cell Disease Treatment: Co-Creation and Implementation of a Preference Exercise With Patients and Caregivers to Inform Drug Development.

Involving patients as co-leaders and co-creators in research is key to reflecting the patient's voice in decision-making. However, co-creation of patient-centered data to inform decisions is rare, especially in early drug development where patient input is critical to prioritizing patient-relevant outcomes and endpoints for use in clinical trials. Despite the industry's growing commitment to patient centricity, most patients are excluded from sharing their expertise in research; more inclusive methods of engaging patients as research partners are needed. We describe a collaboration between a pharmaceutical company and a patient organization in co-leading and co-creating a program to understand priorities of patients and caregivers for treatment features and outcomes in sickle cell disease to inform endpoint selection in clinical development. The results of this program will be used as a basis for continued interaction between patients and the sponsor and to inform ongoing clinical development and evidence-generation activities. This case study demonstrates an approach to meaningful collaborations between patient organizations and pharmaceutical companies aimed at including the patient's voice early in the medical product lifecycle.

The methanogenic redox cofactor F420 is widely synthesized by aerobic soil bacteria.

F420 is a low-potential redox cofactor that mediates the transformations of a wide range of complex organic compounds. Considered one of the rarest cofactors in biology, F420 is best known for its role in methanogenesis and has only been chemically identified in two phyla to date, the Euryarchaeota and Actinobacteria. In this work, we show that this cofactor is more widely distributed than previously reported. We detected the genes encoding all five known F420 biosynthesis enzymes (cofC, cofD, cofE, cofG and cofH) in at least 653 bacterial and 173 archaeal species, including members of the dominant soil phyla Proteobacteria, Chloroflexi and Firmicutes. Metagenome datamining validated that these genes were disproportionately abundant in aerated soils compared with other ecosystems. We confirmed through high-performance liquid chromatography analysis that aerobically grown stationary-phase cultures of three bacterial species, Paracoccus denitrificans, Oligotropha carboxidovorans and Thermomicrobium roseum, synthesized F420, with oligoglutamate sidechains of different lengths. To understand the evolution of F420 biosynthesis, we also analyzed the distribution, phylogeny and genetic organization of the cof genes. Our data suggest that although the Fo precursor to F420 originated in methanogens, F420 itself was first synthesized in an ancestral actinobacterium. F420 biosynthesis genes were then disseminated horizontally to archaea and other bacteria. Together, our findings suggest that the cofactor is more significant in aerobic bacterial metabolism and soil ecosystem composition than previously thought. The cofactor may confer several competitive advantages for aerobic soil bacteria by mediating their central metabolic processes and broadening the range of organic compounds they can synthesize, detoxify and mineralize.

Psychological and physical correlates of musculoskeletal symptoms in male professional divers and offshore workers.

BACKGROUND: Underwater divers are more likely to complain of musculoskeletal symptoms than a control population. Accordingly, we conducted a study to determine whether musculoskeletal symptoms reflected observable physical disorder, to ascertain the relationship between symptoms and measures of mood, memory and executive function and to assess any need for future screening. METHODS: A 10% random sample of responders to a prior postal health questionnaire was examined (151 divers, 120 non-diving offshore workers). Participants underwent physical examination and a neuropsychological test battery for memory and executive function. Participants also completed the Hospital Anxiety and Depression Scale for anxiety (HADSa) and depression (HADSd), and questionnaires for physical health-related quality of life (SF36 PCS), mental health-related quality of life (SF36 MCS), memory (Cognitive Failures Questionnaire (CFQ), Prospective and Retrospective Memory Questionnaire (PRMQ)), executive function (dysexecutive syndrome questionnaire (DEX)), musculoskeletal symptoms (MSS) and general unrelated symptom reporting. RESULTS: Of participants with moderate/severe musculoskeletal symptoms, 52% had physical signs, and of participants with no symptoms, 73% had no physical signs. There was no difference in the prevalence of signs or symptoms between groups. Musculoskeletal symptoms were associated with lower SF36 PCS for both groups. In divers, musculoskeletal symptoms were associated with higher general unrelated symptom reporting and poorer scoring for HADSa, PRMQ, CFQ and DEX with scores remaining within the normative range. A positive physical examination was associated with general unrelated symptom reporting in divers. There were no differences in neuropsychological test scores attributable to either group or musculoskeletal symptoms. CONCLUSIONS: Musculoskeletal symptoms were associated with physical signs, but this was not a strong effect. Reporting of musculoskeletal symptoms by the divers studied was also associated with a tendency to report symptoms generally or somatisation, and caution should be exercised regarding their interpretation as an indication of physical disease or their use for health screening.

Cognitive symptoms and welding fume exposure.

BACKGROUND: Prevalence of moderate to severe cognitive symptoms is markedly higher in UK professional divers who have also worked as a welder (28%) than in either divers who have not welded (18%) or offshore workers who have worked neither as a diver nor as a welder (6%). OBJECTIVES: To determine whether cognitive symptoms are related to welding fume exposure or diving. METHODS: Three age-matched groups of male workers were studied using postal questionnaire: professional divers who had worked as a welder (PDW, n = 361), professional welders who had not dived (NDW, n = 352), and offshore oil field workers who had neither dived nor welded (NDNW, n =503). Health-related quality of life was assessed by the Short Form 12 questionnaire (SF12). Cognitive symptomatology was assessed using the Cognitive Failures Questionnaire (CFQ). A single variable for welding fume exposure (mg m(-3) days) was calculated, incorporating welding experience in different environments and using different welding techniques and respiratory protective equipment. The level of fume exposure during hyperbaric welding operations was measured during such work as ambient PM(10) (particles of 10 µm or less). Diving exposure was assessed as the number of dives performed plus the number of days spent working during saturation diving. RESULTS: Questionnaires were returned by 153 PDW, 108 NDW, and 252 NDNW. SF12 scores were the same in all groups and fell within normative values. Mean (95% CI) CFQ scores were higher in PDW [40.3 (37.7-42.9)] than in both NDW [34.6 (31.6-37.7)] and NDNW [32.1 (30.4-33.9)], but the scores in no groups fell outside the normative range. The mean PM(10) exposure during hyperbaric welding operations was 2.58 mg m(-3). The geometric mean mg m(-3) days (95% CI) for welding fume exposure in NDW [33 128 (24 625-44 567) n = 85] was higher than for that in PDW [10 904 (8103-14 673) n = 112]. For PDW the geometric mean (95% CI) diving exposure was 1491 [(1192-1866) n = 94] dives and days in saturation. In the general linear model regression analyses adjusted for age, alcohol consumption, and somatization, there was no signification association of CFQ score with either welding fume exposure (F = 0.072, P = 0.79, n = 152) or diving exposure (F = 0.042, P = 0.84, n = 74). CONCLUSIONS: In conclusion, cognitive sympomatology was not related to retrospectively assessed measures of welding fume exposure or diving experience. In addition, the levels of cognitive symptomatology, even in PDW, did not exceed normative values.

Hearing symptoms and audiometry in professional divers and offshore workers.

AIMS: The aims are to compare hearing loss between professional divers and offshore workers and to study whether hearing loss symptoms reflected physical disorder. A secondary objective was to study total threshold shift assessment as a method of detecting noise-induced hearing loss (NIHL). METHODS: Participants (151 divers and 120 offshore workers) completed a questionnaire for symptoms and screening audiometry. Audiograms were assessed for total threshold shift at 1, 2, 3, 4 and 6 kHz and the prevalence of referral (within population 5th centile) or warning levels (within population 20th centile) of hearing loss. Audiograms were assessed for an NIHL pattern at four levels by two occupational physicians. RESULTS: Hearing loss symptoms were commoner in divers at all levels of hearing loss regardless of differences between groups on audiometry. Hearing loss in offshore workers was within the population age-adjusted norm. Thirteen per cent of divers were within the 5th percentile for threshold shift for the population norm in contrast to 4% of offshore workers and this was predominantly left sided (OR 3.16, 95% CI 1.13-8.93). This difference was lost after adjustment for history of regular exposure to explosion or gunfire. Divers were more likely to have a pattern of severe NIHL on the left (OR 4.61, 95% CI 1.39-15.39, P < 0.05). Approximately 50% of participants with severe NIHL did not have a referral level of hearing loss. CONCLUSIONS: Divers suffer more NIHL than a control population. Current guidance on the assessment of total threshold shift for the detection of significant NIHL was inadequate in the sample studied.

Comparison of CID versus ETD based MS/MS fragmentation for the analysis of protein ubiquitination.

Ubiquitination has emerged as one of the major post-translational modifications that decide on protein fate, targeting, and regulation of protein function. Whereas the ubiquitination of proteins can be monitored with classic biochemical methods, the mapping of modified side chains proves to be challenging. More recently, mass spectrometry has been applied to identify ubiquitinated proteins and also their sites of modification. Typically, liquid chromatography tandem mass spectrometry (LC-MS/MS) based approaches, including collision-induced fragmentation (CID), have been successfully used in the past. However, a potential difficulty arises from the unstable nature of this modification, and also that the isopeptide bond linkage between C-terminal glycine and the N(epsilon) lysyl side chain is susceptible to fragmentation under these conditions. Here we investigate the utility of electron-transfer dissociation (ETD)-based fragmentation to detect ubiquitination sites in proteins. Our results indicate that ETD can provide alternative fragmentation patterns that allow detection of gly-gly-modified lysyl side chains, in particular z+1 fragment ions derived from triply charged precursor ions. We subsequently applied ETD fragmentation-based analysis and detected novel ubiquitination sites on DNA polymerase B1 that were not easily observed using CID. We conclude that ETD can provide significant alternative fragmentation information that complements CID-derived data to improve the coverage when mapping ubiquitination sites in proteins.

Binding studies of nNOS-active amphibian peptides and Ca2+ calmodulin, using negative ion electrospray ionisation mass spectrometry.

Amphibian peptides which inhibit the formation of nitric oxide by neuronal nitric oxide synthase (nNOS) do so by binding to the protein cofactor, Ca2+calmodulin (Ca2+CaM). Complex formation between active peptides and Ca2+CaM has been demonstrated by negative ion electrospray ionisation mass spectrometry using an aqueous ammonium acetate buffer system. In all cases studied, the assemblies are formed with a 1:1:4 calmodulin/peptide/Ca2+ stoichiometry. In contrast, the complex involving the 20-residue binding domain of the plasma Ca2+ pump C20W (LRRGQILWFRGLNRIQTQIK-OH) with CaM has been shown by previous two-dimensional nuclear magnetic resonance (2D NMR) studies to involve complexation of the C-terminal end of CaM. Under identical conditions to those used for the amphibian peptide study, the ESI complex between C20W and CaM shows specific 1:1:2 stoichiometry. Since complex formation with the studied amphibian peptides requires Ca2+CaM to contain its full complement of four Ca2+ ions, this indicates that the amphibian peptides require both ends of the CaM to effect complex formation. Charge-state analysis and an H/D exchange experiment (with caerin 1.8) suggest that complexation involves Ca2+CaM undergoing a conformational change to a more compact structure.

Effect of protein stabilization on charge state distribution in positive- and negative-ion electrospray ionization mass spectra.

Changes in protein conformation are thought to alter charge state distributions observed in electrospray ionization mass spectra (ESI-MS) of proteins. In most cases, this has been demonstrated by unfolding proteins through acidification of the solution. This methodology changes the properties of the solvent so that changes in the ESI-MS charge envelopes from conformational changes are difficult to separate from the effects of changing solvent on the ionization process. A novel strategy is presented enabling comparison of ESI mass spectra of a folded and partially unfolded protein of the same amino acid sequence subjected to the same experimental protocols and conditions. The N-terminal domain of the Escherichia coli DnaB protein was cyclized by in vivo formation of an amide bond between its N- and C-termini. The properties of this stabilized protein were compared with its linear counterpart. When the linear form was unfolded by decreasing pH, a charge envelope at lower m/z appeared consistent with the presence of a population of unfolded protein. This was observed in both positive-ion and negative-ion ESI mass spectra. Under the same conditions, this low m/z envelope was not present in the ESI mass spectrum of the stable cyclized form. The effects of changing the desolvation temperature in the ionization source of the Q-TOF mass spectrometer were also investigated. Increasing the desolvation temperature had little effect on positive-ion ESI mass spectra, but in negative-ion spectra, a charge envelope at lower m/z appeared, consistent with an increase in the abundance of unfolded protein molecules.

The structure and function of a novel glycerophosphodiesterase from Enterobacter aerogenes.

The structure of the glycerophosphodiesterase (GDPD) from Enterobacter aerogenes, GpdQ, has been solved by SAD phasing from the active site metal ions. Structural analysis indicates that GpdQ belongs to the alpha/beta sandwich metallo-phosphoesterase family, rather than the (alpha/beta)(8) barrel GDPD family, suggesting that GpdQ is a structurally novel GDPD. Hexameric GpdQ is generated by interactions between three dimers. The dimers are formed through domain swapping, stabilised by an inter-chain disulfide bond, and beta-sheet extension. The active site contains a binuclear metal centre, with a fully occupied alpha-metal ion site, and partially occupied beta-metal ion site, as revealed by anomalous scattering analysis. Using a combination of TLS refinement and normal mode analysis, the dynamic movement of GpdQ was investigated. This analysis suggests that the hexameric quaternary structure stabilises the base of the dimer, which promotes "breathing" of the active site cleft. Comparison with other metallo-phosphodiesterases shows that although the central, catalytic, domain is highly conserved, many of these enzymes possess structurally unrelated secondary domains located at the entrance of the active site. We suggest that this could be a common structural feature of metallo-phosphodiesterases that constrains substrate specificity, preventing non-specific phosphodiester hydrolysis.

Health status of professional divers and offshore oil industry workers.

AIMS: To compare the health status of UK professional divers and age-matched non-divers and to contrast offshore divers (OSDs) with non-offshore divers (NOSDs). METHODS: A postal survey sent to 2958 male professional divers, registered with the UK Health & Safety Executive (HSE) before 1991, and 2708 men who had worked in the offshore oil industry in 1990-92 (non-divers). The questionnaire addressed lifestyle, occupation and health status. RESULTS: In all, 56% of divers and 51% of non-divers responded. Three per cent of participants reported ill-health retirement or being off-work on sickness benefit with no difference between groups. Divers were less likely to report asthma or hypertension. Health-related quality of life (SF-12) was within normal limits for both groups but the mental component summary was higher in divers who were also less likely to be receiving medical treatment. Divers were more likely than non-divers to report 'forgetfulness or loss of concentration' (18% versus 6%, OR 3.8, 95% CI 2.7-5.3), musculoskeletal symptoms (41% versus 34%, OR 3.8, 95% CI 2.7-5.3) and 'impaired hearing' (16% versus 11%, OR 1.6, 95% CI 1.2-2.0). These differences were attributable to increased symptom reporting in OSDs and were not present for NOSDs, with the exception of cognitive symptomatology which was commoner in both OSDs (22%, OR 4.8, 95% CI 3.4-6.8) and NOSDs (9%, OR 1.9, 95% CI 1.1-3.3) than in non-divers (6%). CONCLUSIONS: There was increased symptom reporting in OSDs. However, there was no evidence to suggest any major impact on long-term health of UK divers who had started their career before 1991.

A chronic pneumothorax and fitness to fly.

According to Boyle's law, as the pressure falls, the volume of gas rises in an inversely proportional manner. This means that during an aircraft flight, the volume of trapped air in gas filled body chambers will increase. As a consequence, it is fairly well established that individuals with an untreated pneumothorax should not participate in commercial flying due to the risk of it enlarging and the possible development of tension. However, whether this also applies to individuals who have a long-standing, clinically stable pneumothorax is uncertain. The following article describes two adult patients each with a chronic pneumothorax who asked whether they would be fit to fly in an aircraft. We outline their histories and subsequent evaluation which consisted of clinical assessment, computed tomographic imaging, a hypoxic challenge test and exposure to a hypoxic hypobaric environment in a decompression chamber.

The N-terminal domain of alphaB-crystallin is protected from proteolysis by bound substrate.

Alpha-crystallin, a major structural protein of the lens can also function as a molecular chaperone by binding to unfolding substrate proteins. We have used a combination of limited proteolysis at low temperature, and mass spectrometry to identify the regions of alpha-crystallin directly involved in binding to the structurally compromised substrate, reduced alpha-lactalbumin. In the presence of trypsin, alpha-crystallin which had been pre-incubated with substrate showed markedly reduced proteolysis at the C-terminus compared with a control, indicating that the bound substrate restricted access of trypsin to R157, the main cleavage site. Chymotrypsin was able to cleave at residues in both the N- and C-terminal domains. In the presence of substrate, alpha-crystallin showed markedly reduced proteolysis at four sites in the N-terminal domain when compared with the control. Minor differences in cleavage were observed within the C-terminal domain suggesting that the N-terminal region of alpha-crystallin contains the major substrate interaction sites.

Multiple oligomeric forms of Escherichia coli DnaB helicase revealed by electrospray ionisation mass spectrometry.

The Escherichia coli DnaB protein (DnaB(6)) is the hexameric helicase that unwinds genomic DNA so it can be copied by the DNA replication machinery. Loading of the helicase onto DNA requires interactions of DnaB(6) with six molecules of its loading partner protein, DnaC. Nano-electrospray ionisation mass spectrometry (nanoESI-MS) of mutant proteins was used to examine the roles of the residues Phe102 (F102) and Asp82 (D82) in the N-terminal domain of DnaB in the assembly of the hexamer. When the proteins were prepared in 1 M ammonium acetate containing magnesium and adenosine triphosphate (ATP) at pH 7.6, both hexameric and heptameric forms of wild-type and F102W, F102E and D82N mutant DnaBs were observed in mass spectra. The spectra of the D82N mutant also showed substantial amounts of a decameric species and small amounts of a dodecamer. In contrast, the F102H DnaB mutant was incapable of forming oligomers of order higher than the hexamer. Thus, although Phe102 is not the only determinant of hexamer assembly, this residue has a role in oligomerisation. NanoESI mass spectra were obtained of mixtures of DnaB(6) with DnaC. The DnaB(6)(DnaC)(6) complex (calculated M(r) 481 164) was observed only when the two proteins were present in equimolar amounts. The data are consistent with cooperative assembly of the complex. ESI mass spectra of mixtures containing DnaC and ATP showed that DnaC slowly hydrolysed ATP to ADP as indicated by ions corresponding to DnaC/ATP and DnaC/ADP complexes. These experiments show that E. coli DnaB can form a heptameric complex and that nanoESI-MS can be used to probe assembly of large (>0.5 MDa) macromolecular complexes.

An estrogen-platinum terpyridine conjugate: DNA and protein binding and cellular delivery.

A platinum metal complex in which terpyridine joins estradiol (via an ethynyl link) to a platinum with a labile ligand (chloride) has been designed, synthesised and its X-ray crystal structure determined. The aim of this work was to link a targeting motif (in this case estrogen) to a metal-based biomolecule recognition unit (the platinum moiety). The target molecule: 17alpha-[4'-ethynyl-2,2':6',2'-terpyridine]-17beta-estradiol platinum(II) chloride (PtEEtpy) has been shown to bind to both human and bovine serum albumin (SA) and to DNA. FTICR mass spectrometry shows that the bimolecular units are in each case linked through coordination to the platinum with displacement of the chloride ligand. Circular dichroism indicates that a termolecular entity involving PtEEtpy, SA and DNA is formed. A range of electrospray mass spectrometry experiments showed that the PtEEtpy complex breaks and forms coordination bonds relatively easily. A whole cell estrogen receptor assay in an estrogen receptor positive cell (MCF-7) confirms binding of both EEtpy and PtEEtpy to the estrogen receptor in cells. The work demonstrates the concept of linking a targeting moiety (in this case estrogen) to a DNA binding agent.

Objective neuropsychological test performance of professional divers reporting a subjective complaint of "forgetfulness or loss of concentration".

OBJECTIVE: This study attempted to determine whether the higher prevalence of reported "forgetfulness or loss of concentration" among professional divers can be confirmed using objective neuropsychological tests. Secondary aims were to qualify the functional nature of the complaints and to ascertain whether reduced performance was linked to diving history. METHODS: In a case-control study, the neuropsychological test performance of divers complaining of moderate or severe "forgetfulness or loss of concentration" was compared with two age-matched control groups reporting no or slight "forgetfulness or loss of concentration" ("nonforgetful" divers and "nonforgetful" nondivers). The group differences were analyzed using a multivariate analysis of co-variance, followed by canonical discriminant function analysis. Altogether 102 divers with a complaint, 100 nonforgetful divers, and 100 nonforgetful nondivers completed the study. RESULTS: The overall neuropsychological performance differed significantly between the groups [Pillai's trace: F(24,484)=2.04, P=0.003]. Verbal memory (Logical Memory and the California Verbal Learning Test), current intelligence (Wechsler Abbreviated Scale of Intelligence), and sustained attention (rapid visual processing) were poorer among the divers with a complaint than among the nonforgetful divers or the nonforgetful nondivers. The tests of memory, but not those of executive function, differentiated the divers with complaints from the two control groups. Mixed gas bounce diving and surface oxygen decompression diving, but not other techniques, were negatively associated with memory performance. CONCLUSIONS: A cognitive complaint of divers was confirmed using objective tests of neuropsychological performance. Memory, rather than executive function, was affected at the group level, but only to a mild degree. The relationships between diving experience and neuropsychological test performance were small and only seen with diving techniques used in the offshore oil and gas industry.

Effect of hypobaric hypoxia, simulating conditions during long-haul air travel, on coagulation, fibrinolysis, platelet function, and endothelial activation.

CONTEXT: The link between long-haul air travel and venous thromboembolism is the subject of continuing debate. It remains unclear whether the reduced cabin pressure and oxygen tension in the airplane cabin create an increased risk compared with seated immobility at ground level. OBJECTIVE: To determine whether hypobaric hypoxia, which may be encountered during air travel, activates hemostasis. DESIGN, SETTING, AND PARTICIPANTS: A single-blind, crossover study, performed in a hypobaric chamber, to assess the effect of an 8-hour seated exposure to hypobaric hypoxia on hemostasis in 73 healthy volunteers, which was conducted in the United Kingdom from September 2003 to November 2005. Participants were screened for factor V Leiden G1691A and prothrombin G20210A mutation and were excluded if they tested positive. Blood was drawn before and after exposure to assess activation of hemostasis. INTERVENTIONS: Individuals were exposed alternately (> or =1 week apart) to hypobaric hypoxia, similar to the conditions of reduced cabin pressure during commercial air travel (equivalent to atmospheric pressure at an altitude of 2438 m), and normobaric normoxia (control condition; equivalent to atmospheric conditions at ground level, circa 70 m above sea level). MAIN OUTCOME MEASURES: Comparative changes in markers of coagulation activation, fibrinolysis, platelet activation, and endothelial cell activation. RESULTS: Changes were observed in some hemostatic markers during the normobaric exposure, attributed to prolonged sitting and circadian variation. However, there were no significant differences between the changes in the hypobaric and the normobaric exposures. For example, the median difference in change between the hypobaric and normobaric exposure was 0 ng/mL for thrombin-antithrombin complex (95% CI, -0.30 to 0.30 ng/mL); -0.02 [corrected] nmol/L for prothrombin fragment 1 + 2 (95% CI, -0.03 to 0.01 nmol/L); 1.38 ng/mL for D-dimer (95% CI, -3.63 to 9.72 ng/mL); and -2.00% for endogenous thrombin potential (95% CI, -4.00% to 1.00%). CONCLUSION: Our findings do not support the hypothesis that hypobaric hypoxia, of the degree that might be encountered during long-haul air travel, is associated with prothrombotic alterations in the hemostatic system in healthy individuals at low risk of venous thromboembolism.

Proteomic dissection of DNA polymerization.

DNA polymerases replicate the genome by associating with a range of other proteins that enable rapid, high-fidelity copying of DNA. This complex of proteins and nucleic acids is termed the replisome. Proteins of the replisome must interact with other networks of proteins, such as those involved in DNA repair. Many of the proteins involved in DNA polymerization and the accessory proteins are known, but the array of proteins they interact with, and the spatial and temporal arrangement of these interactions, are current research topics. Mass spectrometry is a technique that can be used to identify the sites of these interactions and to determine the precise stoichiometries of binding partners in a functional complex. A complete understanding of the macromolecular interactions involved in DNA replication and repair may lead to discovery of new targets for antibiotics against bacteria and biomarkers for diagnosis of diseases, such as cancer, in humans.

Comparison of negative and positive ion electrospray ionization mass spectra of calmodulin and its complex with trifluoperazine.

The protein calmodulin (apoCaM) undergoes a conformational change when it binds calcium. This structure of the protein (Ca4CaM) is a dumbbell-shaped molecule that undergoes a further profound conformational change on binding of the antipsychotic drug trifluoperazine (TFP). Experimental conditions were developed to prepare samples of apoCaM, Ca4CaM and Ca4CaM/TFP that were substantially free of sodium. The effects of the conformational changes of calmodulin on the charge-state distributions observed in positive ion and negative ion electrospray ionization (ESI) mass spectra were examined. Conversion of apoCaM into Ca4CaM was concomitant with a change in the negative ion ESI mass spectrum whereby the 16- ion was the most abundant ion observed for the apo form and the 8- ion was the most abundant for the complex. In contrast, in the positive ion ESI mass spectra of apoCaM and Ca4CaM, the most abundant species in each case was the 8+ ion. When a complex of Ca4CaMwith TFP was prepared, the most abundant species was the 5+ ion. This is consistent with a conformational change of Ca4CaM that rendered some basic sites inaccessible to ionization in the ESI process. Using the same Ca4CaM/TFP mixture, no complex with TFP was observed in negative ion ESI mass spectra. These observations are discussed in the context of the structural changes that are known to occur in calmodulin, and suggestions are made to explain the apparently conflicting data. The results reported here reflect on the validity of using differences in charge-state distributions observed in ESI mass spectra to assess conformational changes in proteins.