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My 90 years have witnessed a basic transformation in the understanding of disease in terms of molecules, largely through the application of new instruments and technologies. The ultimate distillation of what really works at this level—the quantitative measurements that generate clinical insight from specimens like blood—is clinical chemistry. This field has fascinated me for a long time, partly because of my interest in inventing or improving analytical instruments, and partly as an anchor to real-world biology that is frequently missing in academic research. A second thread of interest to me is how successful research gets done, and how to know when a solitary inventor is needed and when it takes an army. Here I recount some personal experiences relevant to these interests, ranging across several fields and in organizations of widely varying scale, all ultimately linked to clinical chemistry and the human proteome. Interdisciplinary R&D has always fascinated me, and my introduction to it occurred in unusual times, during World War II. I was on active duty in the US Navy before Pearl Harbor as a Photographer’s Mate 2nd Class, and was discharged at the war’s end as a Lieutenant (jg) line officer, with zero instruction in between on how to be a naval officer. Despite (or because of) this fortuitous absence of formal tuition, I found that much of the fun and adventure in life lies in the cracks between disciplines, and that these cracks can be wider in large organizations (like a Navy in wartime) than smaller ones. Flying in blimps off the Carolina coast during the height of antisubmarine warfare, it occurred to me that maybe, lacking a bombsight, we couldn’t actually sink a German submarine if we found it. After developing proper instrumentation, I found experimentally this was largely true, and a proper bombsight was developed. …

Background & Aims: Recent data suggest that polymorphisms in the organic cation transporter (OCTN) genes OCTN1 (SLC22A4) and OCTN2 (SLC22A5) represent disease-causing mutations within the IBD5 locus (chromosome 5q31). We investigated associations with disease susceptibility, phenotype, and evidence for epistasis with CARD15 in 679 patients with Crohn’s disease (CD) or ulcerative colitis (UC). Methods: A total of 374 patients with CD, 305 patients with UC, and 294 healthy controls (HCs) were studied. Genotyping for single nucleotide polymorphisms IGR2096, IGR2198, and IGR2230, OCTN1 variant (SLC22A4 1672C→T), and OCTN2 variant (SLC22A5 −207G→C) was performed using the TaqMan system. Results: The IBD5 OCTN1 and OCTN2 polymorphisms were in strong linkage disequilibrium (D′, >0.959). IGR2198 variant allele frequency (49.1% vs 40.8%; P = .0046) and homozygosity (21% vs 14.8%; P = .044) were associated with CD versus HCs. Variant allelic frequency of OCTN1 (53.6% vs 43%; P = .0008) and OCTN2 (56.1% vs 48.4%; P = .0092) polymorphisms and homozygosity for the OCTN1/2-TC haplotype (28.4% vs 16%; P = .0042) were associated with CD versus HCs. IGR2198 homozygosity and TC homozygosity were associated with stricturing/penetrating disease at follow-up (P = .011 and P = .011, respectively) and disease progression (P = .038 and P = .049, respectively) on univariate analysis and with need for surgery on multivariate analysis (P = .016 and P = .004, respectively). In the absence of the IBD5 risk haplotype, no association of OCTN1/2 variants with CD was detected. No associations were seen with UC. Conclusions: The IBD5 locus influences susceptibility, progression, and need for surgery in CD. However, the contribution of OCTN1/2 variants is not independent of the IBD5 haplotype; a causative role for these genes remains plausible but is not yet proven. Further genetic, functional, and expression data are now required.

A method (denoted SISCAPA) for quantitation of peptides in complex digests is described. In the method, anti-peptide antibodies immobilized on 100 nanoliter nanoaffinity columns are used to enrich specific peptides along with spiked stable-isotope-labeled internal standards of the same sequence. Upon elution from the anti-peptide antibody supports, electrospray mass spectrometry is used to quantitate the peptides (natural and labeled). In a series of pilot experiments, tryptic test peptides were chosen for four proteins of human plasma (hemopexin, alpha1 antichymotrypsin, interleukin-6, and tumor necrosis factor-alpha) from a pool of 10,203 in silico tryptic peptide candidates representing 237 known plasma components. Rabbit polyclonal antibodies raised against the chosen peptide sequences were affinity purified and covalently immobilized on POROS supports. Binding and elution from these supports was shown to provide an average 120-fold enrichment of the antigen peptide relative to others, as measured by selected ion monitoring (SIM) or selected reaction monitoring (SRM) electrospray mass spectrometry. The columns could be recycled with little loss in binding capacity, and generated peptide ion current measurements with cycle-to-cycle coefficients of variation near 5%. Anti-peptide antibody enrichment will contribute to increased sensitivity of MS-based assays, particularly for lower abundance proteins in plasma, and may ultimately allow substitution of a rapid bind/elute process for the time-consuming reverse phase separation now used as a prelude to online MS peptide assays. The method appears suitable for rapid generation of assays for defined proteins, and should find application in the validation of diagnostic protein panels in large sample sets.

We propose a system for continuing surveillance of viral pathogens circulating in large human populations. We base this system on the physical isolation of viruses from large pooled samples of human serum and plasma (e.g., discarded specimens from diagnostic laboratories), followed by shotgun sequencing of the resulting genomes. The technology for concentrating virions from 100-L volumes was developed previously at Oak Ridge National Laboratory, and the means for purifying and concentrating virions from volumes in microliters have been developed recently. At the same time, marine virologists have developed efficient methods for concentrating, amplifying, and sequencing complex viral mixtures obtained from the ocean. Given this existing technology base, we believe an integrated, automated, and contained system for surveillance of the human "virome" can be implemented within 1 to 2 years. Such a system could monitor the levels of known viruses in human populations, rapidly detect outbreaks, and systematically discover novel or variant human viruses.

The human plasma proteome holds the promise of a revolution in disease diagnosis and therapeutic monitoring provided that major challenges in proteomics and related disciplines can be addressed. Plasma is not only the primary clinical specimen but also represents the largest and deepest version of the human proteome present in any sample: in addition to the classical "plasma proteins," it contains all tissue proteins (as leakage markers) plus very numerous distinct immunoglobulin sequences, and it has an extraordinary dynamic range in that more than 10 orders of magnitude in concentration separate albumin and the rarest proteins now measured clinically. Although the restricted dynamic range of conventional proteomic technology (two-dimensional gels and mass spectrometry) has limited its contribution to the list of 289 proteins (tabulated here) that have been reported in plasma to date, very recent advances in multidimensional survey techniques promise at least double this number in the near future. Abundant scientific evidence, from proteomics and other disciplines, suggests that among these are proteins whose abundances and structures change in ways indicative of many, if not most, human diseases. Nevertheless, only a handful of proteins are currently used in routine clinical diagnosis, and the rate of introduction of new protein tests approved by the United States Food and Drug Administration (FDA) has paradoxically declined over the last decade to less than one new protein diagnostic marker per year. We speculate on the reasons behind this large discrepancy between the expectations arising from proteomics and the realities of clinical diagnostics and suggest approaches by which protein-disease associations may be more effectively translated into diagnostic tools in the future.

The effort to produce an index of all human proteins (the human protein index, or HPI) began twenty years ago, before the initiation of the human genome program. Because DNA sequencing technology is inherently simpler and more scalable than protein analytical technology, and because the finiteness of genomes invited a spirit of rapid conquest, the notion of genome sequencing has displaced that of protein databases in the minds of most molecular biologists for the last decade. However, now that the human genome sequence is nearing completion, a major realignment is under way that brings proteins back to the center of biological thinking. Using an influx of new and improved protein technologies--from mass spectrometry to re-engineered two-dimensional (2-D) gel systems, the original objectives of the HPI have been expanded and the time frame for its execution radically shortened. Several additional large scale technology efforts flowing from the HPI are also described.

The lack of sensitive, specific, multiplexable assays for most human proteins is the major technical barrier impeding development of candidate biomarkers into clinically useful tests. Recent progress in mass spectrometry-based assays for proteotypic peptides, particularly those with specific affinity peptide enrichment, offers a systematic and economical path to comprehensive quantitative coverage of the human proteome. A complete suite of assays, e.g. two peptides from the protein product of each of the approximately 20,500 human genes (here termed the human Proteome Detection and Quantitation project), would enable rapid and systematic verification of candidate biomarkers and lay a quantitative foundation for subsequent efforts to define the larger universe of splice variants, post-translational modifications, protein-protein interactions, and tissue localization.

Freshwater lakes and ponds present an ecological interface between humans and a variety of host organisms. They are a habitat for the larval stage of many insects and may serve as a medium for intraspecies and interspecies transmission of viruses such as avian influenza A virus. Furthermore, freshwater bodies are already known repositories for disease-causing viruses such as Norwalk Virus, Coxsackievirus, Echovirus, and Adenovirus. While RNA virus populations have been studied in marine environments, to this date there has been very limited analysis of the viral community in freshwater. Here we present a survey of RNA viruses in Lake Needwood, a freshwater lake in Maryland, USA. Our results indicate that just as in studies of other aquatic environments, the majority of nucleic acid sequences recovered did not show any significant similarity to known sequences. The remaining sequences are mainly from viral types with significant similarity to approximately 30 viral families. We speculate that these novel viruses may infect a variety of hosts including plants, insects, fish, domestic animals and humans. Among these viruses we have discovered a previously unknown dsRNA virus closely related to Banna Virus which is responsible for a febrile illness and is endemic to Southeast Asia. Moreover we found multiple viral sequences distantly related to Israeli Acute Paralysis virus which has been implicated in honeybee colony collapse disorder. Our data suggests that due to their direct contact with humans, domestic and wild animals, freshwater ecosystems might serve as repositories of a wide range of viruses (both pathogenic and non-pathogenic) and possibly be involved in the spread of emerging and pandemic diseases.

To increase understanding of the mechanisms causing syncope in patients over the age of 60, hemodynamic and hormonal responses to 60 minutes of 60 degree head-up tilt were examined in 10 patients with recurrent syncope of unknown origin and five controls with no history of syncope. Nine of 10 patients and all five controls experienced orthostatic intolerance on the tilt table. Syncope or pre-syncope occurred later in controls than in those syncope patients who had exact reproduction of their clinical symptoms (median time 52 versus 22 minutes, P = 0.05). Three different mechanisms of orthostatic intolerance were identified in the 14 subjects: (1) vasovagal syncope, n = 9 (sudden hypotension ± bradycardia); (2) dysautonomic syncope, n = 3 (immediate and gradual parallel declines in both systolic and diastolic pressures with blunted increase in heart rate); (3) psychogenic or vestibular reaction, n = 2 (orthostatic intolerance without hemodynamic changes). Vasovagal syncope patients showed a significant increase in plasma norepinephrine from baseline to maximum level during tilt (100 ± 39% increase, P = 0.03) and a subsequent decrease at the time of syncope (30 ± 5% decrease, P = 0.01), while plasma epinephrine increased markedly from baseline to the time of syncope (827 ± 154% increase, P = 0.0003). Dysautonomy syncope patients had lower supine levels of norepinephrine compared to vasovagal syncope patients (182 ± 30 versus 614 ± 146 pg/mL, P = 0.008) and no significant change in norepinephrine over time; epinephrine levels increased significantly less than in vasovagal patients (net change 38 ± 8 versus 189 ± 56 pg/mL, P = 0.008). Psychogenic and vestibular reactions were distinguished by rising levels of norepinephrine at the time of symptoms. The 50% (5 of 10) prevalence of vasovagal reactions found in the syncope patients suggests that vasovagal syncope may be more common among the elderly than previously recognized.

NOD2/caspase recruitment domain (CARD)15 variants are identified in up to 50% of Crohn's disease (CD) patients. Functional variants of toll-like receptor-4 (TLR4) and CD14 genes may also be relevant to disease pathophysiology. We aimed to assess the contribution of NOD2/CARD15, TLR4 and CD14 variants in Scottish and Irish CD patients. In all, 612 patients with well-characterised inflammatory bowel disease (252 Scottish CD, 247 Scottish UC, 113 Irish CD) and 304 controls were genotyped for variants of NOD2/CARD15 (1007fsinsC, G908R, R702W, P268S), TLR4 (A299G) and CD14 (T-159C). Genotype–phenotype analyses were performed. Variant 1007fsinsC (P=0.003) and G908R (P=0.008) but not R702W (P=0.269) alleles were more prevalent in Scottish CD (4.7, 1.8 and 7.1%, respectively) than Scottish control (2.3, 0.3 and 5.4%). CD allelic frequencies were lower than the series from Europe (P

The abundance profile of the human urinary proteome is known to change as a result of diseases or drug toxicities, particularly of those affecting the kidney and the urogenital tract. A consequence of such insults is the ability to identify proteins in urine, which may be useful as quantitative biomarkers. To succeed in discovering them, reproducible urine sample preparation methods and good protein resolution in two-dimensional electrophoresis (2-DE) gels for parallel semiquantitative protein measurements are desirable. Here, we describe a protein fractionation strategy enriching proteins of molecular masses (M(r)) lower than 30 kDa in a fraction separate from larger proteins. The fraction containing proteins with M(r)s higher than 30 kDa was subsequently subjected to immunoaffinity subtraction chromatography removing most of the highly abundant albumin and immunoglobulin G. Following 2-DE display, superior protein spot resolution was observed. Subsequent high-throughput mass spectrometry analysis of ca. 1400 distinct spots using matrix-assisted laser desorption/ionization-time of flight peptide mass fingerprinting and liquid chromatography-electrospray ionization tandem mass spectrometry lead to the successful identification of 30% of the proteins. As expected from high levels of post-translational modifications in most urinary proteins and the presence of proteolytic products, ca. 420 identified spots collapsed into 150 unique protein annotations. Only a third of the proteins identified in this study are described as classical plasma proteins in circulation, which are known to be relatively abundant in urine despite their retention to a large extent in the glomerular blood filtration process. As a proof of principle that our urinary proteome display effort holds promise for biomarker discovery, proteins isolated from the urine of a renal cell carcinoma patient were profiled prior to and after nephrectomy. Particularly, the decrease in abundance of the kininogen 2-DE gel spot train in urine after surgery was striking.

Plasma contains numerous and diverse proteins with existing and potential therapeutic value. Plasma has been used clinically as both a source of purified derivatives for treating diseases such as hemophilia, and as a diagnostic medium. Recent research directed towards mining plasma's true potential takes advantage of state-of-the-art proteomic analytical methods to develop multi-protein, disease-specific biomarker panels to improve the reliability and specificity of diagnostics. Recombinant production and chromatographic purification methods are increasing the yield and safety of traditional plasma derivatives. Emerging cell-based technologies are being applied to discover novel protein activities and identify epitope-specific antibodies that may have clinical promise.

The goal of proteomics is a comprehensive, quantitative description of protein expression and its changes under the influence of biological perturbations such as disease or drug treatment. Quantitative analysis of protein expression data obtained by high-throughput methods has led us to define the concept of “regulatory homology” and use it to begin to elucidate the basic structure of gene expression control in vivo. Such investigations lay the groundwork for construction of comprehensive databases of mechanisms (cataloguing possible biological outcomes), the next logical step after the soon to be completed cataloguing of genes and gene products. Mechanism databases provide a roadmap towards effective therapeutic intervention that is more direct than that offered by conventional genomics approaches.

Proteins implement most biological functions at the molecular level. As one might expect based on this fact, it appears that the altered functional states associated with toxic effects involve changes in the abundance or structure of proteins. Although numerous specific assays exist to measure changes in the abundance of individual proteins, practical limitations have prevented widespread use of multiple protein assays for the global characterization of toxicity. Recent developments in protein analytical technology are rapidly changing this picture. Two-dimensional gel electrophoresis, a technique capable of resolving and quantitating hundreds of proteins simultaneously, is becoming an automated, high-throughput tool. In parallel, techniques have been developed that allow the resulting deluge of protein measurements to be organized into a prototype Molecular Effects Database™ describing xenobiotic effects in rodent liver. This database can detect, classify, and characterize a broad range of liver toxicity mechanisms. It currently contains approximately 10 million protein measurements, including data on the liver effects of 43 compounds, with a further 50 compounds to be added in 1995. Observed effects range from very broad (sex steroids alter levels of 45% of all liver proteins) to very specific (e.g., hepatic hydroxymethyl glutaryl coenzyme A reductase inhibitors). Companion 2-dimensional databases describing rodent brain and kidney have been initiated, as have linkages to the genomic sequence databases. Assimilation of this approach into research and regulatory toxicology poses an interesting challenge—one that is likely to lead to a radically more sophisticated understanding of toxicity and its biological basis.

We have improved upon the reference two-dimensional (2-D) electrophoretic map of rat liver proteins originally published in 1991 (N. L. Anderson et al., Electrophoresis 1991, 12, 907-930). A total of 53 proteins (102 spots) are now identified, many by microsequencing. In most cases, spots cut from wet, Coomassie Blue stained 2-D gels were submitted to internal tryptic digestion [2], and individual peptides, separated by high-performance liquid chromatography (HPLC), were sequenced using a Perkin-Elmer 477A sequenator. Additional spots were identified using specific antibodies.

An updated two-dimensional electrophoretic map of human plasma proteins is presented, together with a complete listing of the individual protein spots, their locations, size and isoelectric points relative to internal charge standards. Forty-nine polypeptide species are identified, many consisting of multiple spots differing in glycosylation or sequence (e.g., immunoglobulins). A further series of 35 as yet uncharacterized proteins is indicated.

A standard two-dimensional (2-D) protein map of Fischer 344 rat liver (F344MST3) is presented, with a tabular listing of more than 1200 protein species. Sodium dodecyl sulfate (SDS) molecular mass and isoelectric point have been established, based on positions of numerous internal standards. This map has been used to connect and compare hundreds of 2-D gels of rat liver samples from a variety of studies, and forms the nucleus of an expanding database describing rat liver proteins and their regulation by various drugs and toxic agents. An example of such a study, involving regulation of cholesterol synthesis by cholesterol-lowering drugs and a high-cholesterol diet, is presented. Since the map has been obtained with a widely used and highly reproducible 2-D gel system (the Iso-Dalt system), it can be directly related to an expanding body of work in other laboratories.

Background Most emerging health threats are of zoonotic origin. For the overwhelming majority, their causative agents are RNA viruses which include but are not limited to HIV, Influenza, SARS, Ebola, Dengue, and Hantavirus. Of increasing importance therefore is a better understanding of global viral diversity to enable better surveillance and prediction of pandemic threats; this will require rapid and flexible methods for complete viral genome sequencing. Results We have adapted the SISPA methodology 123 to genome sequencing of RNA and DNA viruses. We have demonstrated the utility of the method on various types and sources of viruses, obtaining near complete genome sequence of viruses ranging in size from 3,000–15,000 kb with a median depth of coverage of 14.33. We used this technique to generate full viral genome sequence in the presence of host contaminants, using viral preparations from cell culture supernatant, allantoic fluid and fecal matter. Conclusion The method described is of great utility in generating whole genome assemblies for viruses with little or no available sequence information, viruses from greatly divergent families, previously uncharacterized viruses, or to more fully describe mixed viral infections.

Traditionally, human milk proteins are identified largely by reference to bovine milk. Hence, to identify the major proteins in human milk, we subjected human and bovine milk, in parallel, to high-resolution two-dimensional electrophoresis. Isoelectric precipitation at pH 4.6 was our criterion for distinguishing whey proteins from those of the casein complex. The alpha- and beta-caseins were identified on the basis of relative abundance, relative molecular mass, and relative isoelectric points. Kappa casein was identified as a series of four spots, which disappear from bovine skim milk treated with rennin (chymosin; EC 3.4.23.4) during the clotting process. Para kappa-casein does not appear on the standard ISO-DALT pattern after treatment of bovine milk with rennin, but does appear in BASO-DALT pattern, indicating its high isoelectric point. No protein disappeared from ISO-DALT patterns of human milk after rennin treatment, and no new protein comparable to bovine para kappa-casein appeared in the BASO-DALT patterns; this suggests that kappa-casein is absent from human milk. The proteins identified in human milk patterns include the alpha and beta casein families, lactalbumin, albumin, transferrin, IgA, and lactoferrin. Numerous additional proteins seen in patterns for human milk remain to be identified.

Liver proteins of male C57BL/6T mice treated with 0, 50, or 250 mg/kg Aroclor 1254 were analyzed by high-resolution two-dimensional (2-D) electrophoresis. The resulting patterns were processed using a computerized image analysis system and quantitative data selected for a total of 150 protein spots. On the basis of an analysis of liver proteins form five animals in each treatment group, we found 31 proteins that showed quantitative differences attributable to treatment with chlorinated hydrocarbons at a high level of statistical significance. One of the altered proteins appears to be Mitcon:2, a heat-shock sensitive mitochondrial matrix polypeptide; another appears likely to be microsomal cytochrome b5. The results indicate that quantitative 2-D protein mapping may reveal much more detail regarding the in vivo effects of toxic xenobiotics than has previously been available, and thus allow a more informative approach to the testing of toxic compounds.

A protein series common to the urine and prostatic tissue of 16 of 17 patients with prostatic adenocarcinoma has been identified by high-resolution two-dimensional gel electrophoresis. These proteins, designated PCA-1, have a relative molecular mass in sodium dodecyl sulfate of about 40000. Analyses of urines from eight age-matched controls, seven patients with other ty pes of urogenital malignancies, two patients with benign prostatic hyperplasia, and five patients with malignancies not associated with the urogenital system failed to show PCA-1 in the patterns. These preliminary findings suggest that this protein should be systematically investigated as a candidate marker for prostatic adenocarcinoma in man.

A simple and versatile optical technique for viewing discontinuities in the thickness of horizontal isoelectric focusing gels is described. The discontinuities in the gel are reproducible and relate directly to the ampholyte distribution. Characterization of the capture of the discontinuities in the gel is presented as well as a demonstration of how they may used for localization of focused proteins.

The ISO-DALT system of two-dimensional electrophoresis allows high-resolution separations of proteins and protein subunits. However, the conventional isoelectric focusing employed in this system does not give satisfactory resolution of the more basic proteins, such as histones. The BASO-DALT system was designed to obtain improved resolution of these basic proteins in the first dimension. In this system, phosphatidyl choline is used as the solubilization agent, and allows resolution of many low molecular weight basic proteins that were not seem with more conventional detergents. Using the BASO-DALT system, Novikoff hepatoma chromosomal proteins have been analyzed, and the five histones identified.

Using the ISO-DALT system for two-dimensional (2-D) electrophoresis and the TYCHO system for computer analysis of the resulting protein maps, we obtained high quality quantitative protein abundance data from Coomassie Brilliant Bluestained gels of mouse liver samples. High resolution gels allow more than 100 proteins to be measured with coefficients of variation less than 15 %. A comparison of results from two mouse strains (C57BL/6 and BALB/c) and the cross between them (BCF1) shows that a large number of qutive polymorphisms can be detected, and that, as expected, the amount of protein produced in the heterozygote is intermediate between the parental values. The system described is shown to be capable of reliably detecting decreases in protein abundance such as those expected to result from radiation-induced deletion of one copy of a gene. The implications of these results for the study of gene regulation are discussed in relation to applications in genetics, toxicology, and differentiation.

We analyzed mononuclear leukocytes from patients with various human leukemias by high-resolution two-dimensional electrophoresis. Tumor cells of the granulocytic, monocytic, and lymphoid lineages [obtained from chronic granulocytic leukemia in blast transformation, acute monocytic leukemia, and chronic lymphocytic leukemia (CLL), respectively] can be easily recognized by using a series of cell-type marker proteins identified by comparison of fractionated normal cell populations. B and T cell types of CLL could be distinguished, the results correlating well with those obtained by use of monoclonal-antibody staining methods. In two cases representing almost pure B-cells (classical CLL; 0% T, 85% B) and T-cells (cutaneous T-cell leukemia; 77% T, 0% B), 27 of 29 marker proteins showed quantitative B/T differences comparable to those observed in comparisons of normal B-and T-lymphocytes prepared by cell sorting. These results indicate that cells from relatively well-differentiated leukemias show complex patterns of gene expression very similar to those of the corresponding normal cells and strongly support the use of large marker panels in cell-type determination. Less-well-differentiated acute leukemias [such as acute undifferentiated and acute granulocytic (FAB:M1)] appear to yield protein patterns corresponding less closely to recognizable mature cell types, and may show expression of novel proteins related to the state of differentiation.

The specificity of antibodies was employed in the identification of bands separated by acrylamide gel electrophoresis by subtracting the band prior to the electrophoretic separation. This immunosubtraction was accomplished without sacrificing any of the high resolution obtained on gradient polyacrylamide gels. Using purified antibody (IgG) isolated by chromatography, the subtraction was performed in several ways. The utility of this technique was demonstrated on samples of human serum using antibodies against human transferrin, albumin, α2-macroglobulin, and whole human serum. Specific animal proteins added to the human serum samples were not subtracted.

We briefly review the origins of urinary proteins, as these are now understood, and present a scheme for their separations and evaluation, the ISO-DALT system of two-dimensional electrophoresis. Some results are illustrated and discussed.

Using two-dimensional electrophoresis, we have investigated the responses of human cells in culture to heat shock and to various chemical agents producing a similar effect. These treatments result in the induction of increased synthesis of several specific proteins. One (HShock:1, SDS-molecular mass about 65000) is increased by about 350-fold over the amount in untreated cells. Computer analysis of time-course studies indicates, however, that rates of synthesis of various proteins other than the classical "heat shock proteins" are affected, some of these alterations following time courses quite different from the main (HShock) inductions. The heat shock effect is thus much more complicated than previously realized. We purified the HShock:1 protein from heat-shocked human lymphoblastoid cells, and prepared a rabbit antiserum specific for HShock:1 on nitrocellulose two-dimensional gel transfers of total lymphoblastoid cell protein. A survey of mouse tissues shows high concentrations of an HShock:1-like protein in the testis, and human testes also appears to contain substantial (though lower) concentrations. These results are consistent with the hypothesis (derived from the tissue-culture studies) that the heat shock effect is a general response to the need for increased protein catabolism within the cell. Increased concentrations of HShock:1 are also observed in preparations of blood leukocytes collected from patients after surgery, indicating that some types of physiological trauma may induce the heat shock proteins in man. Using the antiHShock:1 antibody in an immunoassay, it will be possible to systematically examine HShock:1 concentrations in plasma and leukocytes, thereby opening up the possibility of a clinical test based for the first time upon an inducible aspect of cellular gene expression.

A general theory of the evolution of interactive surfaces or sites on cell macromolecules is presented based on the assumption that all macromolecules present in cells are covered with reactive groups to which counter sites can theoretically be made. Initially such potentially reactive sites are considered to exist in pairs. Once one member of a pair is incorporated into a cell, its opposite number is forbidden except where it may be employed to advantage for structure formation. As more and more potentially interactive sites appeared in cells during evolution, a limit was approached where one member of each pair was present. Such a limit has been termed a perfect set, and would both define a “self” and recognize foreignness. The role of perfect sets in the development of the immune system, in differentiation, and in so-called random fixation of amino acid sequences is reviewed.

Two-dimensional electrophoresis was first applied to the analysis of muscle proteins in 1976 when the occurrence of multiple forms of actin was discovered. Since that time the technique has become widely accepted as a new approach to studies of myogenesis, muscle differentiation, and muscle pathology. In addition, two-dimensional electrophoresis now is being used to investigate contractile proteins present in nonmuscle cells. This review will discuss, in general, the technique of two-dimensional electrophoresis in polyacrylamide gels which combines isoelectric focusing and sodium dodecyl sulfate electrophoresis. The application of the technique specifically to muscle protein analysis will be discussed through a review of existing literature on two-dimensional electrophoresis of cultured muscle cells and tissue homogenates. Attention will be given to contractile protein heterogeneities such as alpha, beta, and gamma actin and the embryonic forms of myosin light chains, all discovered through the use of two-dimensional electrophoresis. New information concerning gene expression during muscle differentiation revealed by differences in two-dimensional electrophoresis protein patterns and the use of two-dimensional electrophoresis for studying human muscle pathology through analysis of tissue biopsies will also be discussed.

The proteins in human seminal plasma were mapped by high-resolution two-dimensional electrophoresis (ISO-DALT and BASO-DALT systems). When analyzed under dissociating conditions, samples from normal fertile males revealed a pattern of over 200 proteins, ranging in mass from 10 000 to 100 000 daltons. Comparison of the mapped proteins from these males and those who had undergone vasectomy allowed us to identify one series of glycoproteins as missing from the semen from vasectomized individuals. Glycoproteins isolated by affinity chromatography with use of concanavalin A were also mapped. Some of the protein spots were identified either by co-electrophoresis with purified proteins or by the electrophoretic transfer of proteins to nitrocellulose sheets and subsequent detection by immunological procedures. The proteins identified include a number of serum proteins as well as prostatic acid phosphatase and creatine kinase. Proteolytic events shown to occur during the liquefaction of semen that occurs early after collection indicate the importance of carefully controlled collection and preparation methods for clinical evaluation of seminal plasma. Ethylenediaminetetraacetic acid and phenylmethylsulfonyl fluoride inhibit this proteolysis.

We describe here a computer system for the analysis of high-resolution two-dimensional gel-electrophoresis patterns, with some initial applications. The system (called TYCHO) comprises programs for image acquisition, background subtraction and smoothing, spot detection, gaussian spot modeling, and pattern matching and comparison. It is based on a conventional minicomputer, but makes extensive use of a high-speed array processor in the image-processing and -modeling steps. Used in concert with the ISO-DALT two-dimensional electrophoresis system (Anal. Biochem. 85:331-354, 1978), TYCHO allows quantitative measurement of hundreds of proteins in complex biological samples, and constitutes the initial data-reduction system required for work towards a Human Protein Index.

Antisera against human placental proteins were developed in goats and rabbits, using immunoadjuvants and a prolonged injection schedule. The antisera were absorbed with normal serum proteins and then tested in immunodiffusion against normal and pregnancy sera. Two bands of precipitation due to pregnancy antigens were observed in pregnancy sera as early as 18 days after conception. Detection of these antigens has possibilities for application as an early pregnancy test.

A method of testing batches of ampholytes is presented. By using carbamylated charges standards to co-electrophorese with the protein sample in the first-dimension isoelectric focusing gel, one can monitor, after running and staining the second-dimension sodium dodecyl sulfate (SDS) slabs gel, the continuity of the pH gradient. Charge standards can also be used to check the reproducibility of the pH gradient among batches of ampholytes and to modify the new batch with a small amount of a narrow range ampholyte to assure reproducibility of experiments. Ampholytes for comparison were obtained from three major manufacturers.

The feasibility of constructing a human protein index and the data base associated with it is discussed. The index would be used to explore molecular anatomy and pathology of human cells. At present, the index is closely tied to the only technique that provides the requisite resolution: two-dimensional electrophoresis. Mapping of human cells, tissues and body fluids together with the mapping of micro-biopsy samples of tissues from individuals with many different diseases should create a systematic description of disease at the protein level. (JMT)

Most human serum proteins consistently appear as more than one spot when analyzed by high resolution two-dimensional electrophoresis. We have characterized the “micro-patterns” corresponding to several of the principal proteins. Three minor forms of transferrin are observed (one larger than the predominant form and probably a precursor) and each shows carbohydrate heterogeneity similar to the main form. The haptoglobin β-chain exhibits stepwise heterogeneity in both pI (sialic acid variation) and SDS molecular weight (neutral sugar structure attachment), and is shown to give rise to a smaller, likewise heterogeneous product. The two frequent alleles of α2HS glycoprotein are shown to differ predominantly in sialic acid content rather than polypeptide charge, suggesting a sequence difference at a carbohydrate attachment site. All of the expected sources of microheterogeneity (sialic acid, proteolysis, allelism) are observed, plus an SDS molecular weight microheterogeneity probably due to variation in attachment of large neutral sugar structures.

All cellular macromolecules are covered with sites or reactive domains to which binding sites can be made on other macromolecules. It is proposed that molecular selection began during prebiotic evolution when polymers which adhered together in three dimensional arrays sedimented out of solution. Those tending to interact two dimensionally to form membranes would be much less actively removed, those forming linear structures less so, and the soluble molecules remaining would tend to be those whose interactive counterparts had been removed. Concentration of the suspension thus produced would tend to yield vesicles having surface and possibly internal membranes, having some internal linear aggregates, and containing soluble macromolecules. Subsequent evolution would select against new surfaces which interacted strongly with those already present (unless an advantageous structure is formed), and would, through random variation, gradually yield a large collection of surfaces whose counter sites are absent. The limiting number of such sites is termed a perfect set and contains all of the information required to recognize foreignness. It is therefore a “self” at the molecular level.

Human plasma proteins separated by high-resolution two-dimensional electrophoresis have been electrophoretically transferred to sheets of nitrocellulose using a modification of the method of Towbin, Staehelin, and Gordon [8]. Although the proteins have been denatured in sodium dodecyl sulfate and separated into subunits, the nitrocellulose-bound molecules still react with appropriate specific antisera even after storage of the transfer in air at room temperature for 5 months. Of 25 proteins whose location in the pattern had been previously determined, 24 are specifically revealed on transfers of whole plasma patterns by appropriate antiserum. In addition, 6 previously unidentified proteins (prothrombin, C1s, C4γ, C1s inhibitor, Ig J-chain, and a1AP glycoprotein) have been identified in the pattern for the first time using the transfer technique. It therefore seems likely that a large majority of proteins (> 96 % in this study) retain sufficient conformation throughout the analytical procedure (or can regain it easily afterwards) to be recognized immunologically. The transfer technique thus constitutes a generally useful immunological “third-dimension” in the high-resolution separation of proteins. Of three monoclonal antibodies similarly tested, none could detect antigen transferred to nitrocellulose from a two-dimensional gel, while each bound specifically to the appropriate antigen absorbed in native form to the nitrocellulose.

Two-dimensional electrophoresis of proteins and protein subunits, employing isoelectric focusing in the first dimension and electrophoresis in the presence of sodium dodecyl sulfate in the second, yields the highest resolutions currently available. In this paper separations in the second dimension are considered (the so called DALT system). Methods for multiple-parallel casting of gradient gels in slab gel holders are described. The problem of electrical isolation of the ends of the slabs together with continuous cooling of both surfaces of the slab gel holder along their entire length has been achieved by running the gels in a horizontal direction in a three-compartment tank with the holders inserted in insulating septa. In the system described, 10 slabs are run simultaneously. This, however, is not the upper limit of the number of slabs which can be conveniently run in parallel.

Bioengineering has many significant contributions to make to improvement in the delivery of health care. However, when major research and development efforts are involved, great care must be exercised in project choice to avoid as far as possible needless expense. Even those programs which ultimately result in very significant benefits at low cost to the average citizen pass a high-cost barrier which may take years to cross. A major mistake is to make decisions on the basis of too short a time span. Nearly all biophysical measurements, many tests, and some drugs have all at one time been too costly for general use. Then as the value of the advance became appreciated, inexpensive means for achieving the same ends were devised. A number of bioengineering problems are reviewed which appear feasible but which are not now being done, largely for organizational reasons, to illustrate the point that many worthwhile projects exist now. The importance of decision making in bioengineering on future health-care quality and costs is apparent. Much of future work will probably be at the molecular level. Since support is now fragmented to the point where continued advancement is in jeopardy, it is important that problems in the support and organization of bioengineering and biomedical research be brought continually to public attention.

The preparation and use of rat heart whole homogenate as a standard for the sodium dodecyl sulfate (SDS) electrophoresis dimension of two-dimensional electrophoresis is described. By including the rat heart homogenate in the agarose overlay used to hold an isofocusing gel (first dimension) in contact with a slab gel (second dimension), 80 horizontal lines can be superimposed on a two-dimensional electrophoresis pattern. Such an internal standard is useful as a reference marker for the intercomparison of many gels and also, when calibrated, can be used to determine the approximate molecular weight of proteins.

This paper describes information-handling aspects of the TYCHO I analysis system (Clin, Chem. 27: 1807--1820, 1981), which analyzes two-dimensional electrophoresis gels, matches the individual protein spots with those in a reference pattern, and stores various information--including spot measurements, identifications, treatment profiles, set memberships, and comments--in a computerized database. This and additional information such as amino acid composition and cellular localization is then accessible from an interactive program that includes a pictorial user interface and presents much of the data in graphical form. Use of the TYCHO I system is illustrated by examples drawn from analyses of gel patterns from human leukocytes.