Influence of Primary Structure on Fragmentation of Native-Like Proteins by Ultraviolet Photodissociation.

Analysis of native-like protein structures in the gas phase via native mass spectrometry and auxiliary techniques has become a powerful tool for structural biology applications. In combination with ultraviolet photodissociation (UVPD), native top-down mass spectrometry informs backbone flexibility, topology, hydrogen bonding networks, and conformational changes in protein structure. Although it is known that the primary structure affects dissociation of peptides and proteins in the gas phase, its effect on the types and locations of backbone cleavages promoted by UVPD and concomitant influence on structural characterization of native-like proteins is not well understood. Here, trends in the fragmentation of native-like proteins were evaluated by tracking the propensity of 10 fragment types (a, a+1, b, c, x, x+1, y, y-1, Y, and z) in relation to primary structure in a native-top down UVPD data set encompassing >9600 fragment ions. Differing fragmentation trends are reported for the production of distinct fragment types, attributed to a combination of both direct dissociation pathways from excited electronic states and those surmised to involve intramolecular vibrational energy redistribution after internal conversion. The latter pathways were systematically evaluated to evince the role of proton mobility in the generation of "CID-like" fragments through UVPD, providing pertinent insight into the characterization of native-like proteins. Fragmentation trends presented here are envisioned to enhance analysis of the protein higher-order structure or augment scoring algorithms in the high-throughput analysis of intact proteins.

Modeling Anthropogenic and Environmental Influences on Freshwater Harmful Algal Bloom Development Detected by MERIS Over the Central United States.

Human and ecological health have been threatened by the increase of cyanobacteria harmful algal blooms (cyanoHABs) in freshwater systems. Successful mitigation of this risk requires understanding the factors driving cyanoHABs at a broad scale. To inform management priorities and decisions, we employed random forest modeling to identify major cyanoHAB drivers in 369 freshwater lakes distributed across 15 upper Midwest states during the 2011 bloom season (July-October). We used Cyanobacteria Index (CI_cyano)-A remotely sensed product derived from the MEdium Resolution Imaging Spectrometer (MERIS) aboard the European Space Agency's Envisat satellite-as the response variable to obtain variable importance metrics for 75 landscape and lake physiographic predictor variables. Lakes were stratified into high and low elevation categories to further focus CI_cyano variable importance identification by anthropogenic and natural influences. "High elevation" watershed land cover (LC) was primarily forest or natural vegetation, compared with "low elevation" watersheds LC dominated by anthropogenic landscapes (e.g., agriculture and municipalities). We used the top ranked 25 Random Forest variables to create a classification and regression tree (CART) for both low and high elevation lake designations to identify variable thresholds for possible management mitigation. Mean CI_cyano was 3 times larger for "low elevation" lakes than for "high elevation" lakes, with both mean values exceeding the "High" World Health Organization recreational guidance/action level threshold for cyanobacteria (100,000 cells/mL). Agrarian-related variables were prominent across all 369 lakes and low elevation lakes. High elevation lakes showed more influence of lakeside LC than for the low elevation lakes.

Uniting Native Capillary Electrophoresis and Multistage Ultraviolet Photodissociation Mass Spectrometry for Online Separation and Characterization of Escherichia coli Ribosomal Proteins and Protein Complexes.

With an overarching goal of characterizing the structure of every protein within a cell, identifying its interacting partners, and quantifying the dynamics of the states in which it exists, key developments are still necessary to achieve comprehensive native proteomics by mass spectrometry (MS). In practice, much work remains to optimize reliable online separation methods that are compatible with native MS and improve tandem MS (MS/MS) approaches with respect to when and how energy is deposited into proteins of interest. Herein, we utilize native capillary zone electrophoresis coupled with MS to characterize the proteoforms in the Escherichia coli 70S ribosome. The capabilities of 193 nm ultraviolet photodissociation (UVPD) to yield informative backbone sequence ions are compared to those of higher-energy collisional dissociation (HCD). To further improve sequence coverage values, a multistage MS/MS approach is implemented involving front-end collisional activation to disassemble protein complexes into constituent subunits that are subsequently individually isolated and activated by HCD or UVPD. In total, 48 of the 55 known E. coli ribosomal proteins are identified as 84 unique proteoforms, including 22 protein-metal complexes and 10 protein-protein complexes. Additionally, mapping metal-bound holo fragment ions resulting from UVPD of protein-metal complexes offers insight into the metal-binding sites.

Mapping a Conformational Epitope of Hemagglutinin A Using Native Mass Spectrometry and Ultraviolet Photodissociation.

As the importance of effective vaccines and the role of protein therapeutics in the drug industry continue to expand, alternative strategies to characterize protein complexes are needed. Mass spectrometry (MS) in conjunction with enzymatic digestion or chemical probes has been widely used for mapping binding epitopes at the molecular level. However, advances in instrumentation and application of activation methods capable of accessing higher energy dissociation pathways have recently allowed direct analysis of protein complexes. Here we demonstrate a workflow utilizing native MS and ultraviolet photodissociation (UVPD) to map the antigenic determinants of a model antibody-antigen complex involving hemagglutinin (HA), the primary immunogenic antigen of the influenza virus, and the D1 H1-17/H3-14 antibody which has been shown to confer potent protection to lethal infection in mice despite lacking neutralization activity. Comparison of sequence coverages upon UV photoactivation of HA and of the HA·antibody complex indicates the elimination of some sequence ions that originate from backbone cleavages exclusively along the putative epitope regions of HA in the presence of the antibody. Mapping the number of sequence ions covering the HA antigen versus the HA·antibody complex highlights regions with suppressed backbone cleavage and allows elucidation of unknown epitopes. Moreover, examining the observed fragment ion types generated by UVPD demonstrates a loss in diversity exclusively along the antigenic determinants upon MS/MS of the antibody-antigen complex. UVPD-MS shows promise as a method to rapidly map epitope regions along antibody-antigen complexes as novel antibodies are discovered or developed.

Structural Evaluation of Protein/Metal Complexes via Native Electrospray Ultraviolet Photodissociation Mass Spectrometry.

Ultraviolet photodissociation (UVPD) has emerged as a promising tool to characterize proteins with regard to not only their primary sequences and post-translational modifications, but also their tertiary structures. In this study, three metal-binding proteins, Staphylococcal nuclease, azurin, and calmodulin, are used to demonstrate the use of UVPD to elucidate metal-binding regions via comparisons between the fragmentation patterns of apo (metal-free) and holo (metal-bound) proteins. The binding of staphylococcal nuclease to calcium was evaluated, in addition to a series of lanthanide(III) ions which are expected to bind in a similar manner as calcium. On the basis of comparative analysis of the UVPD spectra, the binding region for calcium and the lanthanide ions was determined to extend from residues 40-50, aligning with the known crystal structure. Similar analysis was performed for both azurin (interrogating copper and silver binding) and calmodulin (four calcium binding sites). This work demonstrates the utility of UVPD methods for determining and analyzing the metal binding sites of a variety of classes of proteins.

Accuracy Assessment of NLCD 2011 percent impervious cover for selected USA Metropolitan Areas.

The emergence of high-resolution land cover data has created the opportunity to assess the accuracy of impervious cover (IC) provided by the National Land Cover Database (NLCD). We assessed the accuracy of the 900 m2 NLCD2011 %IC for 18 metropolitan areas throughout the conterminous United States using reference data from 1 m2 land cover data developed as part of the United States Environmental Protection Agency's EnviroAtlas project. Agreement was assessed from two perspectives: 1) sensitivity to the size of the assessment unit used for the comparison, and 2) utility of NLCD %IC to serve as a proxy for high-resolution IC. The former perspective was considered because statistical relationships can be sensitive to assessment unit size and shape, and the latter perspective was considered because high resolution (reference) %IC data are not available nationwide. The utility of NLCD %IC as a proxy for the high resolution data was assessed for seven lattice (square) cell sizes ranging from 1 ha to 200 ha using four EnviroAtlas IC indicators: 1) %IC per 100 ha (1 km2); 2) %IC by Census block group; 3) %IC within a 15 m (radius) of the riparian zone, and; 4) %IC within a 50 m (radius) of the riparian zone. Agreement was quantified as per assessment unit deviation (NLCD %IC - reference %IC) and summarized as Mean Absolute Deviation (MAD) and Mean Deviation (MD) both within and across the 18 metropolitan areas. Ordinary least squares (OLS) regression (y = reference %IC and x = NLCD %IC) was also used to evaluate the quality of the NLCD %IC data. MAD was ≤ 5% for six of the seven lattice cell sizes. MAD was also ≤ 5% for Census block groups > 100 ha and for both riparian units. These results suggest that uncertainty attributable to the measurement of %IC was no greater than the uncertainty related to the effect of IC on aquatic resources that have been derived from studies of aquatic condition (e.g., benthic fauna) over a range of %IC. Overall, agreement was variable from one metropolitan area to the next. Agreement improved as assessment unit size increased and declined as the level of urbanization (NLCD %IC) increased. NLCD %IC tended to underestimate reference %IC overall, but NLCD %IC was sometimes greater than reference %IC in urbanized settings.

Elusive structural changes of New Delhi metallo-ß-lactamase revealed by ultraviolet photodissociation mass spectrometry.

We use mass spectrometry (MS), under denaturing and non-denaturing solution conditions, along with ultraviolet photodissociation (UVPD) to characterize structural variations in New Delhi metallo-ß-lactamase (NDM) upon perturbation by ligands or mutation. Mapping changes in the abundances and distributions of fragment ions enables sensitive detection of structural alterations throughout the protein. Binding of three covalent inhibitors was characterized: a pentafluorphenyl ester, an O-aryloxycarbonyl hydroxamate, and ebselen. The first two inhibitors modify Lys211 and maintain dizinc binding, although the pentafluorophenyl ester is not selective (Lys214 and Lys216 are also modified). Ebselen reacts with the sole Cys (Cys208) and ejects Zn2 from the active site. For each inhibitor, native UVPD-MS enabled simultaneous detection of the closing of a substrate-binding beta-hairpin loop, identification of covalently-modified residue(s), reporting of the metalation state of the enzyme, and in the case of ebselen, observation of the induction of partial disorder in the C-terminus of the protein. Owing to the ability of native UVPD-MS to track structural changes and metalation state with high sensitivity, we further used this method to evaluate the impact of mutations found in NDM clinical variants. Changes introduced by NDM-4 (M154L) and NDM-6 (A233V) are revealed to propagate through separate networks of interactions to direct zinc ligands, and the combination of these two mutations in NDM-15 (M154L, A233V) results in additive as well as additional structural changes. Insight from UVPD-MS helps to elucidate how distant mutations impact zinc affinity in the evolution of this antibiotic resistance determinant. UVPD-MS is a powerful tool capable of simultaneous reporting of ligand binding, conformational changes and metalation state of NDM, revealing structural aspects of ligand recognition and clinical variants that have proven difficult to probe.

Investigation of GTP-dependent dimerization of G12X K-Ras variants using ultraviolet photodissociation mass spectrometry.

Mutations in the GTPase enzyme K-Ras, specifically at codon G12, remain the most common genetic alterations in human cancers. The mechanisms governing activation of downstream signaling pathways and how they relate back to the identity of the mutation have yet to be completely defined. Here we use native mass spectrometry (MS) combined with ultraviolet photodissociation (UVPD) to investigate the impact of three G12X mutations (G12C, G12V, G12S) on the homodimerization of K-Ras as well as heterodimerization with a downstream effector protein, Raf. Electrospray ionization (ESI) was used to transfer complexes of WT or G12X K-Ras bound to guanosine 5'-diphosphate (GDP) or GppNHp (non-hydrolyzable analogue of GTP) into the gas phase. Relative abundances of homo- or hetero-dimer complexes were estimated from ESI-MS spectra. K-Ras + Raf heterocomplexes were activated with UVPD to probe structural changes responsible for observed differences in the amount of heterocomplex formed for each variant. Holo (ligand-bound) fragment ions resulting from photodissociation suggest the G12X mutants bind Raf along the expected effector binding region (ß-interface) but may interact with Raf via an alternative α-interface as well. Variations in backbone cleavage efficiencies during UV photoactivation of each variant were used to relate mutation identity to structural changes that might impact downstream signaling. Specifically, oncogenic upregulation for hydrogen-bonding amino acid substitutions (G12C, G12S) is achieved by stabilizing ß-interface interactions with Raf, while a bulkier, hydrophobic G12V substitution leads to destabilization of this interface and instead increases the proximity of residues along the α-helical bundles. This study deciphers new pieces of the complex puzzle of how different K-Ras mutations exert influence in downstream signaling.

Tyr1 phosphorylation promotes phosphorylation of Ser2 on the C-terminal domain of eukaryotic RNA polymerase II by P-TEFb.

The Positive Transcription Elongation Factor b (P-TEFb) phosphorylates Ser2 residues of the C-terminal domain (CTD) of the largest subunit (RPB1) of RNA polymerase II and is essential for the transition from transcription initiation to elongation in vivo. Surprisingly, P-TEFb exhibits Ser5 phosphorylation activity in vitro. The mechanism garnering Ser2 specificity to P-TEFb remains elusive and hinders understanding of the transition from transcription initiation to elongation. Through in vitro reconstruction of CTD phosphorylation, mass spectrometry analysis, and chromatin immunoprecipitation sequencing (ChIP-seq) analysis, we uncover a mechanism by which Tyr1 phosphorylation directs the kinase activity of P-TEFb and alters its specificity from Ser5 to Ser2. The loss of Tyr1 phosphorylation causes an accumulation of RNA polymerase II in the promoter region as detected by ChIP-seq. We demonstrate the ability of Tyr1 phosphorylation to generate a heterogeneous CTD modification landscape that expands the CTD's coding potential. These findings provide direct experimental evidence for a combinatorial CTD phosphorylation code wherein previously installed modifications direct the identity and abundance of subsequent coding events by influencing the behavior of downstream enzymes.

Structural and Functional Studies of a gem-Dimethylating Methyltransferase from a trans-Acyltransferase Assembly Line.

The methyl substituents in products of trans-acyltransferase assembly lines are usually incorporated by S-adenosyl-methionine (SAM)-dependent methyltransferase (MT) domains. The gem-dimethyl moieties within the polyketide disorazol are installed through the iterative action of an MT in the third module of its assembly line. The 1.75-Å-resolution crystal structure of this MT helps elucidate how it catalyzes the addition of two methyl groups. Activity assays of point mutants on ß-ketoacyl chains linked to an acyl carrier protein and N-acetylcysteamine provide additional insights into the roles of active site residues. The replacement of an alanine with a phenylalanine at an apparent gatekeeping position resulted in more monomethylation than dimethylation. MTs may form an interface with ketoreductases (KRs) and even mediate the docking of trans-acyltransferase assembly line polypeptides through this association.

Multistage Ultraviolet Photodissociation Mass Spectrometry To Characterize Single Amino Acid Variants of Human Mitochondrial BCAT2.

Unraveling disease mechanisms requires a comprehensive understanding of how the interplay between higher-order structure and protein-ligand interactions impacts the function of a given protein. Recent advances in native mass spectrometry (MS) involving multimodal or higher-energy activation methods have allowed direct interrogation of intact protein complexes in the gas phase, allowing analysis of both composition and subunit connectivity. We report a multistage approach combining collisional activation and 193 nm ultraviolet photodissociation (UVPD) to characterize single amino acid variants of the human mitochondrial enzyme branched-chain amino acid transferase 2 (BCAT2), a protein implicated in chemotherapeutic resistance in glioblastoma tumors. Native electrospray ionization confirms that both proteins exist as homodimers. Front-end collisional activation disassembles the dimers into monomeric subunits that are further interrogated using UVPD to yield high sequence coverage of the mutated region. Additionally, holo (ligand-bound) fragment ions resulting from photodissociation reveal that the mutation causes destabilization of the interactions with a bound cofactor. This study demonstrates the unique advantages of implementing UVPD in a multistage MS approach for analyzing intact protein assemblies.

Tracking the Catalytic Cycle of Adenylate Kinase by Ultraviolet Photodissociation Mass Spectrometry.

The complex interplay of dynamic protein plasticity and specific side-chain interactions with substrate molecules that allows enzymes to catalyze reactions has yet to be fully unraveled. Top-down ultraviolet photodissociation (UVPD) mass spectrometry is used to track snapshots of conformational fluctuations in the phosphotransferase adenylate kinase (AK) throughout its active reaction cycle by characterization of complexes containing AK and each of four different adenosine phosphate ligands. Variations in efficiencies of UVPD backbone cleavages were consistently observed for three α-helices and the adenosine binding regions for AK complexes representing different steps of the catalytic cycle, implying that these stretches of the protein sample various structural microstates as the enzyme undergoes global open-to-closed transitions. Focusing on the conformational impact of recruiting or releasing the Mg2+ cofactor highlights two loop regions for which fragmentation increases upon UVPD, signaling an increase in loop flexibility as the metal cation disrupts the loop interactions with the substrate ligands. Additionally, the observation of holo ions and variations in UVPD backbone cleavage efficiency at R138 implicate this conserved active site residue in stabilizing the donor phosphoryl group during catalysis. This study showcases the utility of UVPD-MS to provide insight into conformational fluctuations of single residues for active enzymes.

Probing the Interaction of Aspergillomarasmine A with Metallo-ß-lactamases NDM-1, VIM-2, and IMP-7.

Metallo-ß-lactamases (MBLs) are a growing threat to the continued efficacy of ß-lactam antibiotics. Recently, aspergillomarasmine A (AMA) was identified as an MBL inhibitor, but the mode of inhibition was not fully characterized. Equilibrium dialysis and metal analysis studies revealed that 2 equiv of AMA effectively removes 1 equiv of Zn(II) from MBLs NDM-1, VIM-2, and IMP-7 when the MBL is at micromolar concentrations. Conversely, 1H NMR studies revealed that 2 equiv of AMA remove 2 equiv of Co(II) from Co(II)-substituted NDM-1, VIM-2, and IMP-7 when the MBL/AMA are at millimolar concentrations. Our findings reveal that AMA inhibits the MBLs by removal of the active site metal ions required for ß-lactam hydrolysis among the most clinically significant MBLs.

Structural heterogeneity in the intrinsically disordered RNA polymerase II C-terminal domain.

RNA polymerase II contains a repetitive, intrinsically disordered, C-terminal domain (CTD) composed of heptads of the consensus sequence YSPTSPS. The CTD is heavily phosphorylated and serves as a scaffold, interacting with factors involved in transcription initiation, elongation and termination, RNA processing and chromatin modification. Despite being a nexus of eukaryotic gene regulation, the structure of the CTD and the structural implications of phosphorylation are poorly understood. Here we present a biophysical and biochemical interrogation of the structure of the full length CTD of Drosophila melanogaster, which we conclude is a compact random coil. Surprisingly, we find that the repetitive CTD is structurally heterogeneous. Phosphorylation causes increases in radius, protein accessibility and stiffness, without disrupting local structural heterogeneity. Additionally, we show the human CTD is also structurally heterogeneous and able to substitute for the D. melanogaster CTD in supporting fly development to adulthood. This finding implicates conserved structural organization, not a precise array of heptad motifs, as important to CTD function.

SITS Derivatization of Peptides to Enhance 266 nm Ultraviolet Photodissociation (UVPD).

N-terminal derivatization of peptides with the chromogenic reagent 4-acetamido-4-isothiocyanatostilbene-2,2-disulfonic acid (SITS) is demonstrated to enhance the efficiency of 266 nm ultraviolet photodissociation (UVPD). Attachment of the chromophore results in a mass shift of 454 Da and provides significant gains in the number and abundances of diagnostic fragment ions upon UVPD. Activation of SITS-tagged peptides with 266 nm UVPD leads to many fragment ions akin to the a/b/y ions commonly produced by CID, along with other sequence ions (c, x, and z) typically accessed through higher energy pathways. Extreme bias towards C-terminal fragment ions is observed upon activation of SITS-tagged peptides using multiple 266 nm laser pulses. Due to the high reaction efficiency of the isothiocyanate coupling to the N-terminus of peptides, we demonstrate the ability to adapt this strategy to a high-throughput LC-MS/MS workflow with 266 nm UVPD. Graphical Abstract ᅟ.

Impact of G12 Mutations on the Structure of K-Ras Probed by Ultraviolet Photodissociation Mass Spectrometry.

Single-residue mutations at Gly12 (G12X) in the GTP-ase protein K-Ras can lead to activation of different downstream signaling pathways, depending on the identity of the mutation, through a poorly defined mechanism. Herein, native mass spectrometry combined with top-down ultraviolet photodissociation (UVPD) was employed to investigate the structural changes occurring from G12X mutations of K-Ras. Complexes between K-Ras or the G12X mutants and guanosine 5'-diphosphate (GDP) or GDPnP (a stable GTP analogue) were transferred to the gas phase by nano-electrospray ionization and characterized using UVPD. Variations in the efficiencies of backbone cleavages were observed upon substitution of GDPnP for GDP as well as for the G12X mutants relative to wild-type K-Ras. An increase in the fragmentation efficiency in the segment containing the first 50 residues was observed for the K-Ras/GDPnP complexes relative to the K-Ras/GDP complexes, whereas a decrease in fragmentation efficiency occurred in the segment containing the last 100 residues. Within these general regions, the specific residues at which changes in fragmentation efficiency occurred correspond to the phosphate and guanine binding regions, respectively, and are indicative of a change in the binding motif upon replacement of the ligand (GDP versus GDPnP). Notably, unique changes in UVPD were observed for each G12X mutant with the cysteine and serine mutations exhibiting similar UVPD changes whereas the valine mutation was significantly different. These findings suggest a mechanism that links the identity of the G12X substitution to different downstream effects through long-range conformational or dynamic effects as detected by variations in UVPD fragmentation.

α-Methylation follows condensation in the gephyronic acid modular polyketide synthase.

C-methyltransferases (MTs) from modular polyketide synthase assembly lines are relatively rare and unexplored domains that are responsible for installing α-methyl groups into nascent polyketide backbones. The stage at which these synthase-embedded enzymes operate during polyketide biosynthesis has yet to be conclusively demonstrated. In this work we establish the activity and substrate preference for six MTs from the gephyronic acid polyketide synthase and demonstrate their ability to methylate both N-acetylcysteamine- and acyl carrier protein-linked ß-ketoacylthioester substrates but not malonyl thioester equivalents. These data strongly indicate that MT-catalyzed methylation occurs immediately downstream of ketosynthase-mediated condensation during polyketide assembly. This work represents the first successful report of MT-catalyzed mono- and dimethylation of simple thioester substrates and provides the groundwork for future mechanistic and engineering studies on this important but poorly understood enzymatic domain.

Linking land cover and water quality in New York City's water supply watersheds.

The Catskill/Delaware reservoirs supply 90% of New York City's drinking water. The City has implemented a series of watershed protection measures, including land acquisition, aimed at preserving water quality in the Catskill/Delaware watersheds. The objective of this study was to examine how relationships between landscape and surface water measurements change between years. Thirty-two drainage areas delineated from surface water sample points (total nitrogen, total phosphorus, and fecal coliform bacteria concentrations) were used in step-wise regression analyses to test landscape and surface-water quality relationships. Two measurements of land use, percent agriculture and percent urban development, were positively related to water quality and consistently present in all regression models. Together these two land uses explained 25 to 75% of the regression model variation. However, the contribution of agriculture to water quality condition showed a decreasing trend with time as overall agricultural land cover decreased. Results from this study demonstrate that relationships between land cover and surface water concentrations of total nitrogen, total phosphorus, and fecal coliform bacteria counts over a large area can be evaluated using a relatively simple geographic information system method. Land managers may find this method useful for targeting resources in relation to a particular water quality concern, focusing best management efforts, and maximizing benefits to water quality with minimal costs.

X-linked thrombocytopenia caused by a novel mutation of GATA-1.

A family with recessive X-linked thrombocytopenia affecting 4 males in 2 generations, characterized by macrothrombocytopenia, profound bleeding, and mild dyserythropoiesis, is described. Microsatellite linkage analysis identified a region of the X chromosome including the GATA-1 gene, which encodes a critical transcription factor involved in erythrocyte and megakaryocyte development. By sequencing the entire coding region of GATA-1, a 2-base mutation was detected that results in a single amino acid substitution (glycine 208 to serine) within a highly conserved portion of the N-terminal zinc finger domain. Restriction fragment length polymorphism confirmed that this novel mutation segregated with the affected males and female carrier. Although not required for DNA binding, Gly208 of GATA-1 is involved in direct interaction with Friend of GATA-1 (FOG), a cofactor required for normal megakaryocytic and erythroid development. These results demonstrate that the GATA-1-FOG interaction is partially disrupted by the mutation and that the greatest effect involves contact with the FOG zinc finger 9. These findings help describe a novel mutation of GATA-1 in humans as a cause of X-linked thrombocytopenia, and they confirm the vital role played by this transcription factor during in vivo megakaryocyte development.

Differential diagnosis and management of an infant presenting in shock with a history of sickle cell anemia and a recent fall.

The presentation of shock in an infant can be subtle, yet must be recognized and treated very quickly to prevent decompensation and cardiopulmonary arrest. Treatment must begin as soon as shock is noted and before or along with the evaluation to establish the etiology of the shock. This case report illustrates these principles by describing an infant with sickle cell anemia who presented to the emergency department in shock after sustaining a fall.