Saliva identification in forensic samples by automated microextraction and intact mass analysis of statherin.

The enzyme α-amylase has long been a commonly targeted protein in serological tests for saliva. While being especially abundant in saliva, α-amylase is detectable in vaginal secretions, sweat, fecal matter, breast milk and other matrices. As a result, assays for α-amylase only provide a presumptive indication of saliva. The availability of mass spectrometry-based tools for the detection of less abundant, but more specific, protein targets (e.g., human statherin) has enabled the development of high confidence assays for human saliva. Sample throughput, however, has traditionally been low due to multi-step workflows for protein extraction, quantitation, enzymatic digestion, solid phase cleanup, and nano-/capillary-based chromatography. Here, we present two novel "direct" single-stage extraction strategies for sample preparation. These feature immunoaffinity purification and reversed-phase solid-phase microextraction in conjunction with intact mass analysis of human statherin for saliva identification. Mass analysis was performed on the Thermo Scientific Q-Exactive™ Orbitrap mass spectrometer with a 10-min analytical run time. Data analysis was performed using Byos® from Protein Metrics. Two sample sets were analyzed with a population of 20 individuals to evaluate detection reliability. A series of casework-type samples were then assayed to evaluate performance in an authentic forensic context. Statherin was confidently identified in 92% and 71% of samples extracted using the immunoaffinity purification and solid phase microextraction approaches, respectively. Overall, immunoaffinity purification outperformed the solid phase microextraction, especially with complex mixtures. In toto, robotic extraction and intact mass spectrometry enable the reliable identification of trace human saliva in a variety of sample types.

High-throughput seminal fluid identification by automated immunoaffinity mass spectrometry.

The identification of semen during a criminal investigation may be a critical component in the prosecution of a sexual assault. Commonly employed enzymatic and affinity-based methods for detection lack specificity, are time-consuming, and only provide a presumptive indication that semen is present where microscopic visualization is unable to meet the throughput demands. Contrary to traditional approaches, protein mass spectrometry provides true confirmatory results, but multiday sample preparation and nanoflow sample separation requirements have limited the practical applicability of these approaches. Aiming at streamlining sexual assault screening by mass spectrometry, the work here coupled a 60-minute rapid tryptic digestion, semenogelin-II peptide affinity purification on an Agilent AssayMap Bravo automation platform, and a 3-minute targeted LC-MS/MS method on an Agilent 6495 triple quadrupole mass spectrometer operating in multiple reaction monitoring mode for detecting semenogelin-II peptides in sexual assault samples. The developed assay was assessed using casework-type samples and was successful in detecting trace levels (0.0001 µl) of semen recovered from both cotton and vaginal swabs, as well as semen recovered from vaginal swabs during menses or adulterated with personal lubricants. This work represents a promising technique for high-throughput seminal fluid identification in sexual assault-type samples by mass spectrometry.

Developmental validation of a multiplex proteomic assay for the identification of forensically relevant biological fluids.

The aim of this study was to validate a multiplex proteomic assay for the identification of high-specificity protein biomarkers by multiple reaction monitoring mass spectrometry on a triple quadrupole mass spectrometer for the accurate, reliable, and confirmatory identification of bodily fluids commonly encountered in a forensic context. This includes the identification of peripheral blood, semen, saliva, urine, and vaginal/menstrual fluid. The assay is able to efficiently identify pure or mixed stains through the identification of target peptide fragments originating from tissue-specific proteins including: uromodulin from urine; prostatic acid phosphatase, prostate specific antigen and semenogelin-II for semen; statherin, submaxillary gland androgen-regulated protein 3B and amylase for saliva; cornulin, martrigel-induced gene C4 protein, suprabasin and neutrophil gelatinase-associated lipocalin for vaginal/menstrual fluid; and alpha-1 antitrypsin, hemopexin, and hemoglobin subunit beta for peripheral blood. Based on the results of the developmental validation studies which included an assessment of reproducibility and repeatability, sensitivity, species specificity, carryover, mixtures, as well as a series of casework type samples. Only a small selection of case samples was unable to unambiguously identify the target fluid including urine recovered from substrates as well as semen when mixed with personal lubricants. Overall, the mass spectrometry-based workflow offers significant advantages compared to existing serological methods.

Direct seminal fluid identification by protease-free high-resolution mass spectrometry.

Serological screening of sexual assault evidence has traditionally focused on enzyme activity and immunochromatographic assays that provide only a presumptive indication of seminal fluid and have limited sensitivity relative to DNA testing. Seminal fluid detection based on protein mass spectrometry represents a "Next Gen" serological technology that overcomes the specificity and sensitivity limitations of traditional serological screening but requires time-consuming sample preparation protocols. This paper describes a novel "peptidomics" approach to seminal fluid detection that eliminates the need for lengthy trypsin digestion. This streamlines sample preparation to a one-step process followed by high-resolution mass spectrometry to identify naturally occurring seminal fluid peptides and low-molecular weight proteins. Multiple protein biomarkers of seminal fluid were consistently and confidently identified based on the multiplexed detection of numerous endogenous peptides. These included Semenogelin I and II (90% and 86% sequence coverage, respectively); Prostate Specific Antigen/p30 (29% sequence coverage); and Prostatic Acid Phosphatase (24% sequence coverage). The performance of this streamlined peptidomics approach to seminal fluid identification in a forensic context was also assessed using simulated casework samples of the type typically collected as part of a sexual assault examination (e.g., oral and vaginal swabs stained with semen). The resulting data demonstrate that sub-microliter quantities of seminal fluid on cotton swabs can be recovered and reliably detected. This supports the forensic applicability of a peptidomic assay for seminal fluid identification with same-day sample preparation and analysis. Future development and streamlined multiplex peptidomic assays for additional biological stains can easily be envisaged.

Effects of organic acids and common household products on the occurrence of false positive test results using immunochromatographic assays.

Forensic serological analyses often rely on lateral flow immunochromatographic assays to detect proteins that are characteristic of forensically relevant body fluids. In this study, we demonstrate that a positive result, however, is not limited to target protein binding. Citric and lactic acids at various pH levels were tested using 9 different commercial immunochromatographic assays. Varying rates of false positive results were observed with commercial serological assays irrespective of brand or target biological fluid over a wide pH range. The use of kit specific buffers were only partially effective in mitigating the occurrence of organic acid-associated false positive results. Common household products containing organic acids were also tested and found to produce non-specific binding events. This is not to suggest that immunochromatographic assays are not useful as presumptive indicators of bodily fluids. Rather, this study provides a cautionary demonstration of the ease with which organic acids in common household products can generate false positives results. This finding underscores the presumptive nature of these antibody-based lateral flow assay systems.

Verification of protein biomarker specificity for the identification of biological stains by quadrupole time-of-flight mass spectrometry.

Advances in proteomics technology over the past decade offer forensic serologists a greatly improved opportunity to accurately characterize the tissue source from which a DNA profile has been developed. Such information can provide critical context to evidence and can help to prioritize downstream DNA analyses. Previous proteome studies compiled panels of "candidate biomarkers" specific to each of five body fluids (i.e., peripheral blood, vaginal/menstrual fluid, seminal fluid, urine, and saliva). Here, a multiplex quadrupole time-of-flight mass spectrometry assay has been developed in order to verify the tissue/body fluid specificity the 23 protein biomarkers that comprise these panels and the consistency with which they can be detected across a sample population of 50 humans. Single-source samples of these human body fluids were accurately identified by the detection of one or more high-specificity biomarkers. Recovery of body fluid samples from a variety of substrates did not impede accurate characterization and, of the potential inhibitors assayed, only chewing tobacco juice appeared to preclude the identification of a target body fluid. Using a series of 2-component mixtures of human body fluids, the multiplex assay accurately identified both components in a single-pass. Only in the case of saliva and peripheral blood did matrix effects appear to impede the detection of salivary proteins.

Discovery of highly specific protein markers for the identification of biological stains.

DNA profiling has transformed the field of forensic biology by making it possible to individualize biological stains. The identification of the stain itself, however, continues to present forensic serologists with significant challenges. Current antibody- and enzyme activity-based assays yield only presumptive results as detection in nontarget body fluids or cross-reactivity with nonhuman sources have both been well documented. For other critical body fluids such as vaginal and menstrual fluids, there are no commercial tests at all. Using a three-pronged, comparative proteomic strategy based on proteome fractionation by HPLC followed by MS, a panel of 29 candidate protein biomarkers have been proposed as highly specific indicators of human saliva, urine, seminal fluid, vaginal fluid, peripheral blood, and menstrual fluid. The combination of consistent identification by multiple strategies in the current study; confirmation in independently compiled proteomic databases; and information on tissue expression and/or functionality from the proteomic literature all support the proposition that these proteins will have utility as reliable biomarkers of their target body fluids. The identification of candidate high-specificity protein biomarkers for human body fluids encountered in forensic investigations lays the foundation for the development of faster and more reliable approaches to the serological analysis of evidentiary stains.

Evolution of gnathostome prodynorphin and proenkephalin: characterization of a shark proenkephalin and prodynorphin cDNAs.

Analyses of prodynorphin and proenkephalin cDNAs cloned from the central nervous system of the shark, Heterodontus portusjacksoni, provided additional evidence that these two opioid precursor-coding genes were most likely directly derived from a common ancestral gene. The two cDNAs could be aligned by inserting only seven gaps. The prodynorphin cDNA encodes five opioid sequences which could be aligned to opioid positions B through F in the proenkephalin cDNA. The sequence identity within the opioid positions was 59% at the amino acid level. Shark α-neo-endorphin, dynorphin A, and dynorphin B have amino acid motifs in common with shark met-enkephalin-8, and shark proenkephalin opioid positions E and F, respectively, which have not been observed in other gnathostome prodynorphin and proenkephalin precursor sequences. Shark prodynorphin encodes both kappa (α-neo-endorphin, dynorphin A, and dynorphin B) and delta (met-enkephalin and leu-enkephalin) opioid sequences. Mixed function prodynorphin precursors (encoding both enkephalins and dynorphins) are also found in representatives of the teleost fishes, lungfishes, and amphibians. It appears that only mammals evolved a prodynorphin precursor that exclusively encodes kappa opioid agonists (dynorphins).

Comparative analysis of the HV1 and HV2 regions of human mitochondrial DNA by denaturing high-performance liquid chromatography.

Denaturing high-performance liquid chromatography (DHPLC) was evaluated as a sequencing-independent means of detecting the presence of sequence differences in pair-wise mixtures of nonconcordant amplicons of human mitochondrial DNA (mtDNA). A total of 920 pair-wise combinations of HV1 and HV2 mtDNA amplicons from 95 individuals were assayed by DHPLC for sequence concordance/nonconcordance. For the 72 combinations of amplicons from different individuals who shared identical DNA sequences, DHPLC assays consistently indicated sequence concordance between the samples. This was in 100% agreement with sequencing data. For the 849 combinations of amplicons which differed in sequence, DHPLC detected the presence of sequence nonconcordance in all but 13 assays to yield 98.5% concordance with sequencing. Thus, DHPLC can be used to detect a diversity of sequence differences (transitions, transversions, insertions, and deletions) in the mtDNA D-loop. Accordingly, DHPLC may have utility as a presumptive indicator of mtDNA sequence concordance samples, as a screen for heteroplasmy/situational mixtures, and as a means for the physical fractionation of the individual contributors to an mtDNA mixture prior to sequencing.

Seasonal changes in CRF-I and urotensin I transcript levels in masu salmon: correlation with cortisol secretion during spawning.

Pacific salmon employ a semelparous reproductive strategy where sexual maturation is followed by rapid senescence and death. Cortisol overproduction has been implicated as the central physiologic event responsible for the post-spawning demise of these fish. Cortisol homeostasis is regulated through the action of hormones of the hypothalamus-pituitary-interrenal (HPI) axis. These include corticotropin-releasing factor (CRF) and urotensin-I (UI). In the present study, masu salmon (Oncorhynchus masou) were assayed for changes in the levels CRF-I and UI mRNA transcripts by quantitative real-time PCR (qRT-PCR). These results were compared to plasma cortisol levels in juvenile, adult, and spawning masu salmon to identify specific regulatory factors that appear to be functionally associated with changes in cortisol levels. Intramuscular implantation of GnRH analog (GnRHa) capsules was also used to determine whether GnRH influences stress hormone levels. In both male and female masu salmon, spawning fish experienced a 5- to 7-fold increase in plasma cortisol levels relative to juvenile non-spawning salmon. Changes in CRF-I mRNA levels were characterized by 1-2 distinctive short-term surges in adult masu salmon. Conversely, seasonal changes in UI mRNA levels displayed broad and sustained increases during the pre-spawning and spawning periods. The increases in UI mRNA levels were positively correlated (R(2)=0.21 male and 0.26 female, p<0.0001) with levels of plasma cortisol in the pre-spawning and spawning periods. Despite the importance of GnRH in sexual maturation and reproduction, the administration of GnRHa to test animals failed to produce broad changes in CRF-I, UI or plasma cortisol levels. These findings suggest a more direct role for UI than for CRF-I in the regulation of cortisol levels in spawning Pacific salmon.

Validation of reduced-scale reactions for the Quantifiler Human DNA kit.

Accurate quantification of DNA samples is an important step in obtaining accurate and reproducible short tandem repeat (STR) profiles. Quantitative real-time-PCR has improved the speed and accuracy of DNA quantification over earlier methods, albeit at significantly greater cost per reaction. Here, the performance of reduced volume (10 microL) DNA quantification assays using the Quantifiler Human DNA Quantification Kit was evaluated using commercial standards and single source biological stains (e.g., venous blood, saliva, and semen). In addition, casework-type samples including those subjected to environmental contaminants containing PCR inhibitors and samples having undergone extensive DNA degradation were also quantified. The concentration of DNA in various forensic samples ranged from 0 to 2.9 ng/microL depending on sample source and/or environmental insult. Compared to full-scale reactions, reduced volume assays displayed equivalent to improved amplification efficiency and sample-to-sample reproducibility (+/-0.01-0.17 C(T FAM)). Furthermore, the use of data from reduced-scale Quantifiler reactions facilitated the accurate determination of the amount of sample DNA extract needed to generate quality STR profiles. The use of 10 microL-scale Quantifiler reaction volumes has the practical benefit of increasing the effective number of reactions per kit by 250%; thereby reducing the cost per assay by 60% while consuming less sample. This is particularly advantageous in cases of consumptive testing.

Resolving mtDNA mixtures by denaturing high-performance liquid chromatography and linkage phase determination.

Mitochondrial DNA (mtDNA) sequencing can provide crucial information to forensic investigators when the quantity and quality of DNA would otherwise be limiting. The difficulty of analyzing mtDNA mixtures, however, has been a significant obstacle to its broader use in forensics. Denaturing high-performance liquid chromatography (DHPLC) in combination with direct sequencing makes it possible to determine the linkage phase of individual amplicons in a mixture. The reliability of the approach is based, in part, on the strong correlation between a change in the relative quantities of different DNA amplicons in a given mixture versus a change in the relative electrophoretic peak heights at mixed base positions. Using standard operating procedures previously validated for use in forensic laboratories, this approach enables sequence-specific fractionation of natural (heteroplasmic) or situational (multi-contributor) DNA mixtures prior to direct sequencing. As a novel approach for the rapid and accurate analysis of DNA mixtures, DHPLC may aid criminal investigators by making it possible to obtain definitive mitochondrial DNA results from otherwise challenging samples.

Separating human DNA mixtures using denaturing high-performance liquid chromatography.

DNA mixtures represent challenging samples that are rarely amenable to direct DNA sequence analysis and many of the strategies available to separate mixtures are both labor and time intensive. Denaturing high-performance liquid chromatography is an accurate and rapid approach for the detection and scoring of mutations. It can also be used to separate DNA mixtures. The technique relies on the chromatographic separation of crosshybridization products to isolate the individual components of a mixture. By eliminating secondary amplification and excessive manipulation prior to sequencing, denaturing high-performance liquid chromatography can streamline the analysis of conditions ranging from somatic mosaicism, microchimerism and mitochondrial heteroplasmy to evidentiary material containing mixtures of DNA encountered in forensic investigations.

Characterizing a proopiomelanocortin cDNA cloned from the brain of the Bichir, Polypterus senegalus: evaluating phylogenetic relationships among ray-finned fish.

There is general agreement that the polypteriform fishes, like Polypterus senegalus, constitute a unique lineage in the evolution of the vertebrates. However, the precise position of these fishes had been a point of controversy since the time of Darwin and Huxley. There is now consensus that the polypteriform fishes are members of superorder Actinopterygii. However, within the Actinopterygii, it is still debatable as to whether the polypteriform fishes are an early offshoot of the Actinopterygii or a more recent sister group to the sturgeon and other extant chondrostean fishes. In this study the sequence of proopiomelanocortin (POMC), the common precursor for the melanocortins and beta-endorphin, was used to evaluate the phylogenetic position of the polypteriform fishes relative to other bony fishes. 3(')RACE and 5(')RACE protocols were used to amplify overlapping regions of a POMC cDNA from the brain of P. senegalus. The full-length POMC cDNA had an open reading frame that encoded 259 amino acids. As seen in most gnathostomes, P. senegalus POMC has three melanocortin sequences (ACTH/alpha-MSH, gamma-MSH, and beta-MSH), and a beta-endorphin region. For phylogenetic analysis, the following POMC sequences were aligned at the amino acid level and analyzed using a maximum parsimony algorithm: P. senegalus, dogfish, sturgeon A, paddlefish A, sockeye salmon A, tilapia, and gar. The dogfish POMC sequence was used as the out-group. In this analysis the P. senegalus POMC sequence formed a clade with the chondrostean POMC sequences (sturgeon A and paddlefish A), and not with the neopterygian sequences (sockeye salmon A, tilapia, and gar). P. senegalus POMC is remarkably similar to sturgeon POMC A. In particular, in both precursors there is evidence for degeneration at the proteolytic cleavage site that precedes the gamma-MSH sequence. Based on the analysis of this nuclear gene it would appear that P. senegalus belongs to a branch of the chrondrostean lineage rather than representing a lineage of ray-finned fish that is ancestral to the chondrostean and neoptyergian ray-finned fishes. Alternatively, if the polypteriform fishes are in fact an early offshoot of the Actinopterygii (the traditional view), then the observations made for P. senegalus POMC relative to the chondrostean POMC sequences is the result of convergence.

Clinical applications of denaturing high-performance liquid chromatography-based genotyping.

AIM: To develop and evaluate heteroduplex forming templates (HFTs) as a common set of molecular standards for genotyping by denaturing high-performance liquid chromatography (DHPLC) using hypervariable regions of human mitochondrial DNA (mtDNA) as a model system. METHODS: Hypervariable regions 1 and 2 from the mtDNA D-loop of 22 maternally related and unrelated human volunteers were amplified by polymerase chain reaction (PCR) and individually mixed with each of three HFTs. Following denaturation and reannealing of the mixture, the resulting hetero- and homoduplicies were separated by DHPLC using temperature-modulated heteroduplex analysis. RESULTS: Each of three HFTs, when cross-hybridized with a target mtDNA amplicon, induced the formation of an assemblage hetero- and homoduplex peaks, which were uniquely characteristic of a given mtDNA sequence variant. The mtDNA DHPLC profiles obtained in the current study were identical between maternal relatives and different between unrelated individuals--consistent with uniparental maternal inheritance of mtDNA in humans. CONCLUSION: DHPLC in combination with a common set of HFTs targeted to a locus of interest can be used as a reliable means of genotyping. DHPLC profiles can be readily stored as a bit-coded string of hetero- and homoduplex peak retention times to form a searchable database. This approach to DHPLC genotyping will have immediate utility in extended pedigree analyses, where it will allow rapid sorting and/or confirmation of maternal lineages. Additional applications of DHPLC profiling include the discovery and scoring of clinically relevant nuclear and mitochondrial loci.

Presence of the delta-MSH sequence in a proopiomelanocortin cDNA cloned from the pituitary of the galeoid shark, Heterodontus portusjacksoni.

Since a fourth MSH sequence, delta-MSH, has been detected in the proopiomelanocortin (POMC) gene of a dogfish and a stingray, members of superorder Squalea (class Chondrichthyes), it is possible that this novel MSH sequence might be a feature common to the POMC genes of all modern sharks and rays. As an initial step towards addressing this question, a full-length POMC cDNA was cloned and sequenced from the pituitary of the Port Jackson shark, Heterodontus portusjacksoni. The Port Jackson shark represents one of the oldest lineages in superorder Galea, and this superorder together with superorder Squalea form infraclass Neoselachii (the extant sharks and rays). The Port Jackson shark POMC cDNA has an open reading frame that is 1032 nucleotides in length and encodes the deduced amino acids sequences for beta-endorphin, ACTH/alpha-MSH, beta-MSH, gamma-MSH, and delta-MSH. Port Jackson shark delta-MSH has 83% primary sequence identity with dogfish and stingray delta-MSH, and it appears that the delta-MSH sequence may have been the result of an internal domain duplication and reinsertion of the beta-MSH sequence. The presence of the delta-MSH sequence in the POMC genes of representatives of both superorders of infraclass Neoselachii would indicate that the delta-MSH sequence must have been present in the ancestral euselachian shark that gave rise to the neoselachian radiation.

Evaluating the radiation of the POMC gene in teleosts: characterization of American eel POMC.

A distinctive feature of the pituitary hormone precursor, proopiomelanocortin (POMC), is the presence of multiple melanocortin core sequences (HFRW), and one copy of the opioid, beta-endorphin. In the older lineages of ray-finned fish (i.e., orders Acipenseriformes and Semionotiformes), certain extant lobe-finned fish (Australian lungfish and African lungfish), and the tetrapods there are three melanocortin regions in POMC: ACTH/alphaMSH, beta-MSH, and gamma-MSH. However, among the teleosts, the most recent radiation of the ray-finned fishes, the gamma-MSH sequence is absent from the POMC genes of euteleosts like the carp, tilapia, chum salmon, sockeye salmon, and rainbow trout. The objective of this study was to determine whether the gamma-MSH sequence still may be present in the POMC gene of a more basal lineage of the teleosts such as a representative from subdivision Elopomorpha. To this end, a POMC cDNA was cloned and sequenced from the pituitary of the American eel, Anguilla rostrata (order Anguilliformes, family Anguillidae). The open reading frame of the eel POMC cDNA was 648 nucleotides in length and encoded 216 amino acids. As predicted, eel POMC contained the deduced amino acid sequences for beta-endorphin, ACTH/alpha-MSH, and beta-MSH. These end-products displayed primary sequence features that are common to ray-finned fish. Eel POMC lacks a gamma-MSH sequence and a large portion of the joining peptide region. In this regard, the eel POMC gene thus displays features very similar to the POMC genes that have been sequenced from euteleosts. Although it is conceivable that the gamma-MSH sequence may be present in representatives from the other basal extant lineages of teleosts (i.e., subdivisions Osteoglossomorpha or Clupeomorpha), it is also possible that the deletion that resulted in the loss of the gamma-MSH sequence occurred in the ancestral neopterygian that gave rise to the teleosts. In this case, the gamma-MSH sequence should be absent in all extant teleosts.

Forensic utility of mitochondrial DNA analysis based on denaturing high-performance liquid chromatography.

AIM: To determine the forensic utility for pairwise DNA comparisons and DNA mixture resolution with denaturing high-performance liquid chromatography (DHPLC) of human mitochondrial DNA (mtDNA). METHODS: MtDNA hypervariable regions (HV) 1 and 2 from the mtDNA D-loop were amplified by the polymerase chain reaction and mixed between known and unknown sample sources. The DNA mixtures were denatured and reannealed, and the resultant homo- and heteroduplices were evaluated by temperature-modulated heteroduplex analysis by the DHPLC method. RESULTS: All 144 pairwise comparisons of HV1 and HV2 mtDNA fragments were successfully resolved by the DHPLC method. Forensic proficiency test standards were successfully resolved and DHPLC match/non-match results agreed with sequencing results provided by the test providers. The DHPLC method successfully identified one questioned sample that was prepared by the test provider as a body fluid mixture. MtDNA amplicon mixtures could be separated into their constitutive components by DHPLC and fraction collection approaches. CONCLUSIONS: DHPLC methods provide the forensic scientist with a powerful tool to rapidly screen mtDNA and may result in standardized methods to resolve mtDNA mixtures. These advances will allow mtDNA analysis in cases not previously examined by current sequencing-based approaches and could allow more forensic case samples to be entered into the proposed mtDNA Combined DNA Index System (CODIS trade mark ) databank as a result of mtDNA mixture resolution.

Analyzing the evolution of the opioid/orphanin gene family.

Advances in molecular biology have made it possible to rapidly obtain the amino acid sequence of neuropeptide precursors-either by cloning and sequencing the cDNA that encodes the precursor, or by reconstructing the arrangement of exons and introns in a neuropeptide-coding gene through genomic approaches. The databases generated from these molecular approaches have been used to design probes to identify the cells that express the gene, or to ascertain the rate of expression of the gene, and even to predict the post-translational modifications that can generate functional neuropeptides from a biologically inert precursor. Although the power of these approaches is substantial, it is appreciated that a gene sequence or an mRNA sequence reflects the potential products that may be assembled in a secretory cell. To understand the functional capabilities of the secretory cell, the molecular genetics approaches must be combined with procedures that actually characterize the end-products generated by the secretory cell. Recent advances in two-dimensional gel electrophoresis and mass spectrometry now make it possible to analyze neuropeptides from a relatively small amount of tissue. These procedures can reveal novel end-products, tissue-specific endoproteolytic cleavage events, and developmental shifts in post-translational processing schemes. A gene family that illustrates all of these processes and the advantages of combining genomics with proteomics is the opioid/orphanin gene family.