Monitoring recombinant protein expression in bacteria by rapid evaporative ionisation mass spectrometry.

RATIONALE: There is increasing interest in methods of direct analysis mass spectrometry that bypass complex sample preparation steps. METHODS: One of the most interesting new ionisation methods is rapid evaporative ionisation mass spectrometry (REIMS) in which samples are vapourised and the combustion products are subsequently ionised and analysed by mass spectrometry (Synapt G2si). The only sample preparation required is the recovery of a cell pellet from a culture that can be analysed immediately. RESULTS: We demonstrate that REIMS can be used to monitor the expression of heterologous recombinant proteins in Escherichia coli. Clear segregation was achievable between bacteria harvesting plasmids that were strongly expressed and other cultures in which the plasmid did not result in the expression of large amounts of recombinant product. CONCLUSIONS: REIMS has considerable potential as a near-instantaneous monitoring tool for protein production in a biotechnology environment.

Individual odour signatures that mice learn are shaped by involatile major urinary proteins (MUPs).

BACKGROUND: Reliable recognition of individuals requires phenotypic identity signatures that are both individually distinctive and appropriately stable over time. Individual-specific vocalisations or visual patterning are well documented among birds and some mammals, whilst odours play a key role in social recognition across many vertebrates and invertebrates. Less well understood, though, is whether individuals are recognised through variation in cues that arise incidentally from a wide variety of genetic and non-genetic differences between individuals, or whether animals evolve distinctive polymorphic signals to advertise identity reliably. As a bioassay to understand the derivation of individual-specific odour signatures, we use female attraction to the individual odours of male house mice (Mus musculus domesticus), learned on contact with a male's scent marks. RESULTS: Learned volatile odour signatures are determined predominantly by individual differences in involatile major urinary protein (MUP) signatures, a specialised set of communication proteins that mice secrete in their urine. Recognition of odour signatures in genetically distinct mice depended on differences in individual MUP genotype. Direct manipulation using recombinant MUPs confirmed predictable changes in volatile signature recognition according to the degree of matching between MUP profiles and the learned urine template. Both the relative amount of the male-specific MUP pheromone darcin, which induces odour learning, and other MUP isoforms influenced learned odour signatures. By contrast, odour recognition was not significantly influenced by individual major histocompatibility complex genotype. MUP profiles shape volatile odour signatures through isoform-specific differences in binding and release of urinary volatiles from scent deposits, such that volatile signatures were recognised from the urinary protein fraction alone. Manipulation using recombinant MUPs led to quantitative changes in the release of known MUP ligands from scent deposits, with MUP-specific and volatile-specific effects. CONCLUSIONS: Despite assumptions that many genes contribute to odours that can be used to recognise individuals, mice have evolved a polymorphic combinatorial MUP signature that shapes distinctive volatile signatures in their scent. Such specific signals may be more prevalent within complex body odours than previously realised, contributing to the evolution of phenotypic diversity within species. However, differences in selection may also result in species-specific constraints on the ability to recognise individuals through complex body scents.

RePLiCal: A QconCAT Protein for Retention Time Standardization in Proteomics Studies.

This study introduces a new reversed-phase liquid chromatography retention time (RT) standard, RePLiCal (Reversed-phase liquid chromatography calibrant), produced using QconCAT technology. The synthetic protein contains 27 lysine-terminating calibrant peptides, meaning that the same complement of standards can be generated using either Lys-C or trypsin-based digestion protocols. RePLiCal was designed such that each constituent peptide is unique with respect to all eukaryotic proteomes, thereby enabling integration into a wide range of proteomic analyses. RePLiCal has been benchmarked against three commercially available peptide RT standard kits and outperforms all in terms of LC gradient coverage. RePLiCal also provides a higher number of calibrant points for chromatographic retention time standardization and normalization. The standard provides stable RTs over long analysis times and can be readily transferred between different LC gradients and nUHPLC instruments. Moreover, RePLiCal can be used to predict RTs for other peptides in a timely manner. Furthermore, it is shown that RePLiCal can be used effectively to evaluate trapping column performance for nUHPLC instruments using trap-elute configurations, to optimize gradients to maximize peptide and protein identification rates, and to recalibrate the m/z scale of mass spectrometry data post-acquisition.

From sexual attraction to maternal aggression: when pheromones change their behavioural significance.

This article is part of a Special Issue "Chemosignals and Reproduction". This paper reviews the role of chemosignals in the socio-sexual interactions of female mice, and reports two experiments testing the role of pup-derived chemosignals and the male sexual pheromone darcin in inducing and promoting maternal aggression. Female mice are attracted to urine-borne male pheromones. Volatile and non-volatile urine fractions have been proposed to contain olfactory and vomeronasal pheromones. In particular, the male-specific major urinary protein (MUP) MUP20, darcin, has been shown to be rewarding and attractive to females. Non-urinary male chemosignals, such as the lacrimal protein ESP1, promote lordosis in female mice, but its attractive properties are still to be tested. There is evidence indicating that ESP1 and MUPs are detected by vomeronasal type 2 receptors (V2R). When a female mouse becomes pregnant, she undergoes dramatic changes in her physiology and behaviour. She builds a nest for her pups and takes care of them. Dams also defend the nest against conspecific intruders, attacking especially gonadally intact males. Maternal behaviour is dependent on a functional olfactory system, thus suggesting a role of chemosignals in the development of maternal behaviour. Our first experiment demonstrates, however, that pup chemosignals are not sufficient to induce maternal aggression in virgin females. In addition, it is known that vomeronasal stimuli are needed for maternal aggression. Since MUPs (and other molecules) are able to promote intermale aggression, in our second experiment we test if the attractive MUP darcin also promotes attacks on castrated male intruders by lactating dams. Our findings demonstrate that the same chemosignal, darcin, promotes attraction or aggression according to female reproductive state.

Comparative study of the molecular variation between 'central' and 'peripheral' MUPs and significance for behavioural signalling.

MUPs (major urinary proteins) play an important role in chemical signalling in rodents and possibly other animals. In the house mouse (Mus musculus domesticus) MUPs in urine and other bodily fluids trigger a range of behavioural responses that are only partially understood. There are at least 21 Mup genes in the C57BL/6 mouse genome, all located on chromosome 4, encoding sequences of high similarity. Further analysis separates the MUPs into two groups, the 'central' near-identical MUPs with over 97% sequence identity and the 'peripheral' MUPs with a greater degree of heterogeneity and approximately 20-30% non-conserved amino acids. This review focuses on differences between the two MUP sub-groups and categorizes these changes in terms of molecular structure and pheromone binding. As small differences in amino acid sequence can result in marked changes in behavioural response to the signal, we explore the potential of single amino acid changes to affect chemical signalling and protein stabilization. Using analysis of existing molecular structures available in the PDB we compare the chemical and physical properties of the ligand cavities between the MUPs. Furthermore, we identify differences on the solvent exposed surfaces of the proteins, which are characteristic of protein-protein interaction sites. Correlations can be seen between molecular heterogeneity and the specialized roles attributed to some MUPs.

Outcomes of labor induction at 39 weeks in pregnancies with a prior cesarean delivery.

BACKGROUND: The optimal timing of induction for those undergoing a trial of labor after cesarean section has not been established. The little data which supports the consideration of induction at 39 weeks gestation excludes those with a history of prior cesarean section. OBJECTIVE: To determine the risks and benefits of elective induction of labor (IOL) at 39 weeks compared with expectant management (EM) until 42 weeks in pregnancies complicated by one previous cesarean delivery. STUDY DESIGN: This is a retrospective cohort analysis of singleton non-anomalous pregnancies in the United States between January 2015 and December 2017. Data was provided by the CDC National Center for Health Statistics, Division of Vital Statistics. Analyses included only pregnancies with a history of one previous cesarean delivery (CD). Perinatal outcomes of pregnancies electively induced at 39 weeks (IOL) were compared to pregnancies that were induced, augmented or underwent spontaneous labor between 40 and 42 weeks (EM). Unlabored cesarean deliveries were excluded. Outcomes of interest included: cesarean delivery, intra-amniotic infection, blood transfusion, adult intensive care unit (ICU) admission, uterine rupture, hysterectomy, 5-minute Apgar score ≤3, prolonged neonatal ventilation, neonatal ICU (NICU) admission, neonatal seizure, perinatal/neonatal death. Log-binomial regression analysis was performed to calculate the relative risk (RR) for each outcome of interest, adjusting for confounding variables. RESULTS: There were 50,136 pregnancies included for analysis with 9,381 women in the IOL group. Compared with EM, IOL at 39 weeks decreased the risk of intra-amniotic infection (1.7% vs 3.0%, p < .001; aRR: 0.58, 95% CI: [0.49-0.68]), blood transfusion (0.3% vs. 0.5%, p = .03; aRR: 0.66, 95% CI: [0.45-0.98]), and low 5-minute Apgar score (0.31% vs 0.47%, p = .031; aRR: 0.66, 95% CI: [0.44-0.97]). Conversely, IOL increased the risk of cesarean delivery (49.0% vs 27.6%, p < .001; aRR: 1.72, 95% CI: [1.68-1.77]). Furthermore, in the EM group, 919 pregnancies developed preeclampsia and 42 progressed to eclampsia. There were no differences in other perinatal outcomes. CONCLUSION: In pregnancies complicated by one previous cesarean delivery, elective induction of labor at 39 weeks reduced the risk of intra-amniotic infection, blood transfusion, and low 5-minute Apgar score while increased the risk of repeat cesarean delivery.

Darcin: a male pheromone that stimulates female memory and sexual attraction to an individual male's odour.

BACKGROUND: Among invertebrates, specific pheromones elicit inherent (fixed) behavioural responses to coordinate social behaviours such as sexual recognition and attraction. By contrast, the much more complex social odours of mammals provide a broad range of information about the individual owner and stimulate individual-specific responses that are modulated by learning. How do mammals use such odours to coordinate important social interactions such as sexual attraction while allowing for individual-specific choice? We hypothesized that male mouse urine contains a specific pheromonal component that invokes inherent sexual attraction to the scent and which also stimulates female memory and conditions sexual attraction to the airborne odours of an individual scent owner associated with this pheromone. RESULTS: Using wild-stock house mice to ensure natural responses that generalize across individual genomes, we identify a single atypical male-specific major urinary protein (MUP) of mass 18893Da that invokes a female's inherent sexual attraction to male compared to female urinary scent. Attraction to this protein pheromone, which we named darcin, was as strong as the attraction to intact male urine. Importantly, contact with darcin also stimulated a strong learned attraction to the associated airborne urinary odour of an individual male, such that, subsequently, females were attracted to the airborne scent of that specific individual but not to that of other males. CONCLUSIONS: This involatile protein is a mammalian male sex pheromone that stimulates a flexible response to individual-specific odours through associative learning and memory, allowing female sexual attraction to be inherent but selective towards particular males. This 'darcin effect' offers a new system to investigate the neural basis of individual-specific memories in the brain and give new insights into the regulation of behaviour in complex social mammals.See associated Commentary http://www.biomedcentral.com/1741-7007/8/71.

Large-scale and significant expression from pseudogenes in Sodalis glossinidius - a facultative bacterial endosymbiont.

The majority of bacterial genomes have high coding efficiencies, but there are some genomes of intracellular bacteria that have low gene density. The genome of the endosymbiont Sodalis glossinidius contains almost 50 % pseudogenes containing mutations that putatively silence them at the genomic level. We have applied multiple 'omic' strategies, combining Illumina and Pacific Biosciences Single-Molecule Real-Time DNA sequencing and annotation, stranded RNA sequencing and proteome analysis to better understand the transcriptional and translational landscape of Sodalis pseudogenes, and potential mechanisms for their control. Between 53 and 74 % of the Sodalis transcriptome remains active in cell-free culture. The mean sense transcription from coding domain sequences (CDSs) is four times greater than that from pseudogenes. Comparative genomic analysis of six Illumina-sequenced Sodalis isolates from different host Glossina species shows pseudogenes make up ~40 % of the 2729 genes in the core genome, suggesting that they are stable and/or that Sodalis is a recent introduction across the genus Glossina as a facultative symbiont. These data shed further light on the importance of transcriptional and translational control in deciphering host-microbe interactions. The combination of genomics, transcriptomics and proteomics gives a multidimensional perspective for studying prokaryotic genomes with a view to elucidating evolutionary adaptation to novel environmental niches.

Molecular complexity of the major urinary protein system of the Norway rat, Rattus norvegicus.

Major urinary proteins (MUP) are the major component of the urinary protein fraction in house mice (Mus spp.) and rats (Rattus spp.). The structure, polymorphism and functions of these lipocalins have been well described in the western European house mouse (Mus musculus domesticus), clarifying their role in semiochemical communication. The complexity of these roles in the mouse raises the question of similar functions in other rodents, including the Norway rat, Rattus norvegicus. Norway rats express MUPs in urine but information about specific MUP isoform sequences and functions is limited. In this study, we present a detailed molecular characterization of the MUP proteoforms expressed in the urine of two laboratory strains, Wistar Han and Brown Norway, and wild caught animals, using a combination of manual gene annotation, intact protein mass spectrometry and bottom-up mass spectrometry-based proteomic approaches. Cluster analysis shows the existence of only 10 predicted mup genes. Further, detailed sequencing of the urinary MUP isoforms reveals a less complex pattern of primary sequence polymorphism in the rat than the mouse. However, unlike the mouse, rat MUPs exhibit added complexity in the form of post-translational modifications, including the phosphorylation of Ser4 in some isoforms, and exoproteolytic trimming of specific isoforms. Our results raise the possibility that urinary MUPs may have different roles in rat chemical communication than those they play in the house mouse. Shotgun proteomics data are available via ProteomExchange with identifier PXD013986.

Is there value for serial ultrasonographic assessment of cervical lengths after a cerclage?

OBJECTIVE: The objective of the study was to determine the value of serial ultrasonographic cervical length (CL) measurements after cerclage to predict preterm delivery. STUDY DESIGN: Retrospective ultrasonographic and outcome data from singleton pregnancies with cerclage were reviewed. Using transvaginal ultrasound (TVS), overall CL obtained before cerclage placement, 2 weeks after cerclage, and before delivery were compared between women who delivered preterm (less than 37 weeks) and term. The overall CL including CL above (CLA) and below the cerclage (CLB) were compared using the SAS program. RESULTS: Cerclage was placed at 15.7 +/- 3.6 weeks (mean +/- SD) in 57 women. The overall CL before cerclage, 2 weeks after cerclage, and the last TVS before delivery was not different in preterm and term births. The odds ratio of a measurable CLA for preterm delivery by TVS was 0.87 (0.78 to 0.95, 95% confidence interval). Thirty-two patients (56%) had absent CLA at 26.7 +/- 4.4 weeks. Of these, 16 (50%) were delivered for preterm premature rupture of membranes (PPROM) and chorioamnionitis (sensitivity of 100%, specificity of 61%, positive predictive value of 50%, and negative predictive value of 100%). CONCLUSION: Although the overall cervical length by serial TVS after cerclage did not predict preterm birth, absent CLA is associated with preterm delivery, chorioamnionitis, and PPROM.

Breed associations for canine exocrine pancreatic insufficiency.

BACKGROUND: Knowledge of breed associations is valuable to clinicians and researchers investigating diseases with a genetic basis. HYPOTHESIS: Among symptomatic dogs tested for exocrine pancreatic insufficiency (EPI) by canine trypsin-like immunoreactivity (cTLI) assay, EPI is common in certain breeds and rare in others. Some breeds may be overrepresented or underrepresented in the population of dogs with EPI. Pathogenesis of EPI may be different among breeds. ANIMALS: Client-owned dogs with clinical signs, tested for EPI by radioimmunoassay of serum cTLI, were used. METHODS: In this retrospective study, results of 13,069 cTLI assays were reviewed. RESULTS: An association with EPI was found in Chows, Cavalier King Charles Spaniels (CKCS), Rough-Coated Collies (RCC), and German Shepherd Dogs (GSD) (all P < .001). Chows (median, 16 months) were younger at diagnosis than CKCS (median, 72 months, P < .001), but not significantly different from GSD (median, 36 months, P = .10) or RCC (median, 36 months, P = .16). GSD (P < .001) and RCC (P = .015) were younger at diagnosis than CKCS. Boxers (P < .001), Golden Retrievers (P < .001), Labrador Retrievers (P < .001), Rottweilers (P = .022), and Weimaraners (P = .002) were underrepresented in the population with EPI. CONCLUSIONS AND CLINICAL IMPLICATIONS: An association with EPI in Chows has not previously been reported. In breeds with early-onset EPI, immune-mediated mechanisms are possible or the disease may be congenital. When EPI manifests later, as in CKCS, pathogenesis is likely different (eg, secondary to chronic pancreatitis). Underrepresentation of certain breeds among dogs with EPI has not previously been recognized and may imply the existence of breed-specific mechanisms that protect pancreatic tissue from injury.

Quantitative Proteomics of Cerebrospinal Fluid in Paediatric Pneumococcal Meningitis.

Streptococcus pneumoniae is responsible for diseases causing major global public health problems, including meningitis, pneumonia and septicaemia. Despite recent advances in antimicrobial therapy, pneumococcal meningitis remains a life-threatening disease. Furthermore, long-term sequelae are a major concern for survivors. Hence, a better understanding of the processes occurring in the central nervous system is crucial to the development of more effective management strategies. We used mass spectrometry based quantitative proteomics to identify protein changes in cerebrospinal fluid from children with Streptococcus pneumoniae infection, compared with children admitted to hospital with bacterial meningitis symptoms but negative diagnosis. Samples were analysed, by label free proteomics, in two independent cohorts (cohort 1: cases (n = 8) and hospital controls (n = 4); cohort 2: cases (n = 8), hospital controls (n = 8)). Over 200 human proteins were differentially expressed in each cohort, of which 65% were common to both. Proteins involved in the immune response and exosome signalling were significantly enriched in the infected samples. For a subset of proteins derived from the proteome analysis, we corroborated the proteomics data in a third cohort (hospital controls (n = 15), healthy controls (n = 5), cases (n = 20)) by automated quantitative western blotting, with excellent agreement with our proteomics findings. Proteomics data are available via ProteomeXchange with identifier PXD004219.

The structure, stability and pheromone binding of the male mouse protein sex pheromone darcin.

Mouse urine contains highly polymorphic major urinary proteins that have multiple functions in scent communication through their abilities to bind, transport and release hydrophobic volatile pheromones. The mouse genome encodes for about 20 of these proteins and are classified, based on amino acid sequence similarity and tissue expression patterns, as either central or peripheral major urinary proteins. Darcin is a male specific peripheral major urinary protein and is distinctive in its role in inherent female attraction. A comparison of the structure and biophysical properties of darcin with MUP11, which belongs to the central class, highlights similarity in the overall structure between the two proteins. The thermodynamic stability, however, differs between the two proteins, with darcin being much more stable. Furthermore, the affinity of a small pheromone mimetic is higher for darcin, although darcin is more discriminatory, being unable to bind bulkier ligands. These attributes are due to the hydrophobic ligand binding cavity of darcin being smaller, caused by the presence of larger amino acid side chains. Thus, the physical and chemical characteristics of the binding cavity, together with its extreme stability, are consistent with darcin being able to exert its function after release into the environment.

Pheromonal induction of spatial learning in mice.

Many mammals use scent marking for sexual and competitive advertisement, but little is known about the mechanism by which scents are used to locate mates and competitors. We show that darcin, an involatile protein sex pheromone in male mouse urine, can rapidly condition preference for its remembered location among females and competitor males so that animals prefer to spend time in the site even when scent is absent. Learned spatial preference is conditioned through contact with darcin in a single trial and remembered for approximately 14 days. This pheromone-induced learning allows animals to relocate sites of particular social relevance and provides proof that pheromones such as darcin can be highly potent stimuli for social learning.

1H, 15N and 13C resonance assignment of darcin, a mouse major urinary protein.

Darcin is an important lipocalin of the urinary MUP family. These beta-barrel structures differ subtly in sequence and function and facilitate communication between members of the mouse population via scent marks. Polymorphism within the family has led to the hypothesis that individual MUPs can also contribute to social and physiological information of the scent owner and thus demonstrates the necessity for structural investigation of these variations. Using conventional triple resonance experiments, (1)H (15)N and (13)C assignment of recombinant N terminal hexa-histidine tagged Darcin has been achieved. The corresponding chemical shifts have been deposited in the BioMagResBank; Accession No. 16840.

Regional variation in parvalbumin isoform expression correlates with muscle performance in common carp (Cyprinus carpio).

The mechanical properties of the axial muscles vary along the length of a fish's body. This variation in performance correlates with the expression of certain muscle proteins. Parvalbumin (PARV) is an important calcium binding protein that helps modulate intracellular calcium levels which set the size and shape of the muscle calcium transient. It therefore has a central role in determining the functional properties of the muscle. Transcript data revealed eight specific isoforms of PARV in common carp (Cyprinus carpio) skeletal muscle which we classified as alpha1 and beta1-7. This study is the first to show expression of all eight skeletal muscle PARV isoforms in carp at the protein level and relate regional differences in expression to performance. All of the PARV isoforms were characterised at the protein level using 2D-PAGE and tandem mass spectrometry. Comparison of carp muscle from different regions of the fish revealed a higher level of expression of PARV isoforms beta4 and beta5 in the anterior region, which was accompanied by an increase in the rate of relaxation. We postulate that changes in specific PARV isoform expression are an important part of the adaptive change in muscle mechanical properties in response to varying functional demands and environmental change.

Characterization of cauxin in the urine of domestic and big cats.

Cauxin is an abundant protein in feline urine. We have used proteomics strategies to characterize cauxin from the urine of domestic cats and a number of big cat species. Proteins were resolved by gel-based electrophoretic purification and subjected to in-gel digestion with trypsin. The resultant tryptic peptides were mass-measured by matrix-assisted laser desorption ionization time of flight mass spectrometry. Peptides were also resolved by liquid chromatography and analyzed by electrospray ionization and tandem mass spectrometry to generate fragment ion data to infer the amino acid sequence. We identified cauxin polymorphisms and corrected a sequencing artifact in cauxin from the domestic cat. The proteomics data also provided positive evidence for the presence of a cauxin homolog in the urine of big cats (Pantherinae), including the Sumatran tiger, Asiatic lion, clouded leopard, Persian leopard, and jaguar. The levels of cauxin in the urine of all big cats were substantially lower than that in the urine of intact male domestic cats.

Anabolic effects of a non-myotoxic dose of the beta2-adrenergic receptor agonist clenbuterol on rat plantaris muscle.

Previous investigations of the effects of clenbuterol have used suprapharmacological doses that induce myocyte death, alter muscle phenotype, and do not approximate the proposed therapeutic dose for humans. Recently, we reported that smaller doses of clenbuterol induce muscle growth without causing myocyte death. In the present study we used histochemical and proteomic techniques to investigate the molecular effects of this dose. Male Wistar rats (n = 6, per group) were infused with saline or 10 microg/kg/day clenbuterol via subcutaneously implanted osmotic pumps. After 14 days the animals' plantaris muscles were isolated for histochemical and proteomic analyses. Clenbuterol induced significant muscle growth with concomitant protein accretion and preferential hypertrophy of fast oxidative glycolytic fibers. Clenbuterol reduced the optical density of mitochondrial staining in fast fibers by 20% and the glycogen content of the muscle by 30%. Differential analysis of two-dimensional gels showed that heat shock protein 72 and beta-enolase increased, whereas aldolase A, phosphogylcerate mutase, and adenylate kinase decreased. Only heat shock protein 72 has previously been investigated in clenbuterol-treated muscles. The clenbuterol-induced increase in muscle growth was concomitant with qualitative changes in the muscle's proteome that need to be considered when proposing therapeutic uses for this agent.

Global cooling: cold acclimation and the expression of soluble proteins in carp skeletal muscle.

The common carp (Cyprinus carpio) has a well-developed capacity to modify muscle properties in response to changes in temperature. Understanding the mechanisms underpinning this phenotypic response at the protein level may provide fundamental insights into the molecular basis of adaptive processes in skeletal muscle. In this study, common carp were subjected to a cooling regimen and soluble extracts of muscle homogenates were separated by 1-D SDS-PAGE and 2-DE. Proteins were identified using MALDI-TOF-MS and de novo peptide sequencing using LC-MS/MS. The 2-D gel was populated with numerous protein spots that were fragments of all three muscle isoforms (M1, M2 and M3) of carp creatine kinase (CK). The accumulation of the CK fragments was enhanced when the carp were cooled to 10 degrees C. The protein changes observed in the skeletal muscle of carp subjected to cold acclimation were compared to changes described in a previous transcript analysis study. Genes encoding CK isoforms were downregulated and the genes encoding key proteins of the ubiquitin-proteasome pathway were upregulated. These findings are consistent with a specific cold-induced enhancement of proteolysis of CK.

A proteome analysis of the subcutaneous gel in avian hatchlings.

An appropriate level of water loss from eggs is critical to successful hatching. This water may be lost from the egg by evaporation, but where water loss is suboptimal, it is commonly observed that the hatchlings contain substantial amounts of a subcutaneous gel-like fluid. To characterize this fluid, we have analyzed the proteins that are contained within it. The protein complement comprised a small number of proteins in high concentrations. Proteomics analysis of the constituent proteins identified virtually all of these abundant proteins and confirmed that the subcutaneous gel was very similar in protein composition to plasma. However, the subcutaneous gel was substantially depleted of fibrinogen. It is possible that activation of the final stages of the coagulation process might account for the enhanced viscosity, creating a gel-like material that is relatively immobile in the subcutaneous space. This gel may function as a water volume that is partitioned during embryonic development in order to mitigate the effects of high water content of the egg caused by low mass loss during incubation and in some instances might also function as a water reserve to support the hatchling in the first few hours of life free of the shell.