Optimization of Data-Dependent Acquisition Parameters for Coupling High-Speed Separations with LC-MS/MS for Protein Identifications

Recent developments in chromatography, such as ultra-HPLC and superficially porous particles, offer significantly improved peptide separation. The narrow peak widths, often only several seconds, can permit a 15-min liquid chromatography run to have a similar peak capacity as a 60-min run using traditional HPLC approaches. In theory, these larger peak capacities should provide higher protein coverage and/or more protein identifications when incorporated into a proteomic workflow. We initially observed a decrease in protein coverage when implementing these faster chromatographic approaches, due to data-dependent acquisition (DDA) settings that were not properly set to match the narrow peak widths resulting from newly implemented, fast separation techniques. Oversampling of high-intensity peptides lead to low protein-sequence coverage, and tandem mass spectra (MS/MS) from lower-intensity peptides were of poor quality, as automated MS/MS events were occurring late on chromatographic peaks. These observations led us to optimize DDA settings to use these fast separations. Optimized DDA settings were applied to the analysis of Trypanosome brucei peptides, yielding peptide identifications at a rate almost five times faster than previously used methodologies. The described approach significantly improves protein identification workflows that use typical available instrumentation.     

Identification of mature nocistatin and nociceptin in human brain and cerebrospinal fluid by mass spectrometry combined with affinity chromatography and HPLC

Nocistatin (NST) and nociceptin/orphanin FQ (NCP) are two important bio-peptides derived from the precursor protein prepronociceptin (ppNCP), involved in several central nervous system (CNS) functions including pain transmission. Since the actual form of human NST in CNS is not fully characterized, we studied the structure of NST from human brain tissue and cerebrospinal fluid (CSF) samples. NST and NCP were isolated from human brain and CSF samples by affinity chromatography combined with HPLC. Mass spectrometry was used for the identification and characterization of the peptides. The total NST immunoreactivity was detected as 11.5+/-2.3 pmol/g tissue for the brain and 0.44 pmol/ml for the pooled CSF sample after the HPLC purification by radioimmunoassay. The presence of two different forms of mature nocistatin (NST-17 and NST-30) and a possible N-terminal methionine cleaved NST-29 were confirmed by both radioimmunoassay and mass spectrometry. Affinity chromatography, HPLC and mass spectrometry methods used in this study were highly sensitive and suitable for identification of actual chemical structures and quantification of very small amounts of peptides in biological samples. The present findings may help further for search for new treatment of neuropathic pain, which is often poorly managed by current therapies.     

Identification of proNeuropeptide FFA peptides processed in neuronal and non-neuronal cells and in nervous tissue.

Peptides which should be generated from the neuropeptide FF (NPFF) precursor were identified in a neuronal (human neuroblastoma SH-SY5Y) cell line and in COS-7 cells after transient transfection of the human proNPFFA cDNA and were compared with those detected in the mouse spinal cord. After reverse-phase high performance liquid chromatography of soluble material, NPFF-related peptides were immunodetected with antisera raised against NPFF and identified by using on-line capillary liquid chromatography/nanospray ion trap tandem mass spectrometry. Neuronal and non-neuronal cells generated different peptides from the same precursor. In addition to NPFF, SQA-NPFF (Ser-Gln-Ala-Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-amide) and NPAF were identified in the human neuroblastoma while only NPFF was clearly identified in COS-7 cells. In mouse, in addition to previously detected NPFF and NPSF, SPA-NPFF (Ser-Pro-Ala-Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-amide), the homologous peptide of SQA-NPFF, were characterized. These data on intracellular processing of proNeuropeptide FFA are discussed in regard to the known enzymatic processing mechanisms.     

Mouse liver NAD(P)H:quinone acceptor oxidoreductase: protein sequence analysis by tandem mass spectrometry, cDNA cloning, expression in Escherichia coli, and enzyme activity analysis.

The amino acid sequence of mouse liver NAD(P)H:quinone acceptor oxidoreductase (EC 1.6.99.2) has been determined by tandem mass spectrometry and deduced from the nucleotide sequence of the cDNA encoding for the enzyme. The electrospray mass spectral analyses revealed, as previously reported (Prochaska HJ, Talalay P, 1986, J Biol Chem 261:1372-1378), that the 2 forms--the hydrophilic and hydrophobic forms--of the mouse liver quinone reductase have the same molecular weight. No amino acid sequence differences were found by tandem mass spectral analyses of tryptic peptides of the 2 forms. Moreover, the amino-termini of the mouse enzymes are acetylated as determined by tandem mass spectrometry. Further, only 1 cDNA species encoding for the quinone reductase was found. These results suggest that the 2 forms of the mouse quinone reductase have the same primary sequences, and that any difference between the 2 forms may be attributed to a labile posttranslational modification. Analysis of the mouse quinone reductase cDNA revealed that the enzyme is 273 amino acids long and has a sequence homologous to those of rat and human quinone reductases. In this study, the mouse quinone reductase cDNA was also ligated into a prokaryotic expression plasmid pKK233.2, and the constructed plasmid was used to transform Escherichia coli strain JM109. The E. coli-expressed mouse quinone reductase was purified and characterized. Although mouse quinone reductase has an amino acid sequence similar to those of the rat and human enzymes, the mouse enzyme has a higher NAD(P)H-menadione reductase activity and is less sensitive to flavones and dicoumarol, 2 known inhibitors of the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparison of methods to examine the endogenous peptides of fetal calf serum

There is a great desire to relate the patterns of endogenous peptides in blood to human disease and drug response. The best practices for the preparation of blood fluids for analysis are not clear and also relatively few of the peptides in blood have been identified by tandem mass spectrometry. We compared a number of sample preparation methods to extract endogenous peptides including C18 reversed phase, precipitation, and ultrafiltration. We examined the results of these sample preparation methods by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) and liquid chromatography-tandem mass spectrometry (MS/MS) using MALDI-TOF/TOF and electrospray ionization-ion trap. Peptides from solid-phase extraction with C18 in the range of hundreds of femtomoles per spot were detected from the equivalent of 1 μL of serum by MALDI-TOF. We observed endogenous serum peptides from fibrinogen α- and β-chain, complement C3, α-2-HS-glycoprotein, albumin, serine (or cysteine) proteinase inhibitor, factor VIII, plasminogen, immunoglobulin, and other abundant blood proteins. However, we also recorded significant MS/MS spectra from tumor necrosis factor-α-, major histocompatibility complex-, and angiotensin-related peptides, as well as peptides from collagens and other low-abundance proteins. Amino acid substitutions were detected and a phosphorylated peptide was also observed. This is the first time the endogenous peptides of fetal serum have been examined by MS and where peptides from low-abundance proteins, phosphopeptides, and amino acid substitutions were detected.     

MALDI-TOF/TOF和LC-MS/MS液质联用分析大鼠脑脉络丛的多肽组

脉络丛(choroid plexus,CP)位于血液与脑脊液(cerebrospinal fluid,CSF)之间,不仅是CSF的重要来源,而且是构成血液-脑脊液屏障(blood-cerebrospinal fluid barrier,BCB)的组织基础.CP参与脑组织中一些血源性多肽的输送以及自身多肽合成的生理过程,在维持脑微环境动态平衡和调节中枢神经系统的正常功能方面起到非常重要的作用.本研究分别运用MALDI-TOF/TOF和LC-MS/MS液质联用系统分析了成年SD大鼠血液-脑脊液屏障(即脉络丛组织)中的多肽组.共鉴定到163个多肽(P0.001),这些多肽为69种蛋白质的降解肽段,其中ATP合酶(ATP synthase),细胞色素c(cytochrome c),血红蛋白(hemoglobin),NADH-辅酶Q氧化还原酶(NADH-ubiquinone oxidoreductase),β珠蛋白(beta-globin)这5种蛋白质的肽段数占总肽段数的50%以上,并且部分多肽序列相似度高,类似其前体蛋白的逐步降解片段,而这些前体蛋白质的分子量多数在10kD至20kD之间.上述研究结果为SD大鼠脉络丛组织的生理功能研究及组织多肽组学的研究方法提供了有价值的科学资料.     

基于质谱和生物信息学分析的小菜蛾蛋白质鉴定

本研究以非模式昆虫小菜蛾Plutella xylostella为材料, 对比2, 3, 4龄幼虫的蛋白质组双向电泳图谱, 得到24个蛋白质差异点, 从中选取了编号为1111的差异表达蛋白质点进行质谱鉴定和生物信息学分析. 采用胶内酶解的多肽进行MALDI-TOF/TOF分析, 获得该点的肽质量指纹图谱（PMF）及串联质谱（MS/MS）图谱。将获得的PMF分别用MASCOT和ProFound等常用软件在NCBInr的Metazoa蛋白质数据库进行搜索, 匹配结果不理想. 进一步用PMF+MS/MS谱图搜索NCBInr的Metazoa蛋白质数据库, 以及小菜蛾EST数据库。 在NCBInr库中匹配结果为拟暗果蝇Drosophila pseudoobscura中的一种假定蛋白GA18218-PA, 而用EST库搜索的结果为家蚕Bombyx mori的ATP合酶的亚基。为验证搜索结果, 将该蛋白质点进行磺基异硫氰酸苯酯（SPITC）化学衍生后de novo测序, 最后确认该点可能为ATP合酶的一个亚基。最后着重讨论了蛋白质的质谱鉴定与生物信息学分析的联合使用, 希望据此选择出最适合于非模式昆虫蛋白质组学鉴定的方法。     

Proteomic analysis of Tityus discrepans scorpion venom and amino acid sequence of novel toxins

The Venezuelan scorpion Tityus discrepans is known to cause human fatalities. We describe the first complete proteomic analysis of its venom. By HPLC 58 different fractions were obtained and 205 different components were identified by MS analysis. Components having molecular masses from 272 to 57 908 amu were found. Forty homogeneous components had their N-terminal amino acid sequence determined by Edman degradation, from which two new peptides named TdK2 and TdK3 (meaning T. discrepans (Td) K(+) channel toxins 2 and 3) were fully characterized. The first contains 34 amino acid residues with a molecular mass of 3451 amu, and the second has 36 amino acids with 3832 amu. Both peptides are tightly bound by three disulfide bridges. TdK2 was shown to block reversibly the Shaker B K(+)-channel expressed heterologously in Sf9 cells. The systematic number assigned to TdK2 is alpha-KTx-18.2 and that of TdK3 is alpha-KTx-18.3. Comparative analysis of the amino acid sequences found suggests that this venom contains peptides highly similar to those that block K(+) channels, as well as those that modify the gating mechanisms of Na(+) channels, found in other scorpions. Additionally, peptides similar to defensins were also identified.     

The Use of Ammonium Formate as a Mobile-Phase Modifier for LC-MS/MS Analysis of Tryptic Digests

A major challenge facing current mass spectrometry (MS)-based proteomics research is the large concentration range displayed in biological systems, which far exceeds the dynamic range of commonly available mass spectrometers. One approach to overcome this limitation is to improve online reversed-phase liquid chromatography (RP-LC) separation methodologies. LC mobile-phase modifiers are used to improve peak shape and increase sample load tolerance. Trifluoroacetic acid (TFA) is a commonly used mobile-phase modifier, as it produces peptide separations that are far superior to other additives. However, TFA leads to signal suppression when incorporated with electrospray ionization (ESI), and thus, other modifiers, such as formic acid (FA), are used for LC-MS applications. FA exhibits significantly less signal suppression, but is not as effective of a modifier as TFA. An alternative mobile-phase modifier is the combination of FA and ammonium formate (AF), which has been shown to improve peptide separations. The ESI-MS compatibility of this modifier has not been investigated, particularly for proteomic applications. This work compares the separation metrics of mobile phases modified with FA and FA/AF and explores the use of FA/AF for the LC-MS analysis of tryptic digests. Standard tryptic-digest peptides were used for comparative analysis of peak capacity and sample load tolerance. The compatibility of FA/AF in proteomic applications was examined with the analysis of soluble proteins from canine prostate carcinoma tissue. Overall, the use of FA/AF improved online RP-LC separations and led to significant increases in peptide identifications with improved protein sequence coverage.     

Comparative proteomic analysis of the insulin‐induced L6 myotube secretome

Emerging evidence has revealed an endocrine function for skeletal muscle; in fact, certain anti‐inflammatory cytokines are secreted only from contractile skeletal muscle. However, the skeletal muscle secretome as a whole is poorly characterized, as is how it changes in response to extracellular stimuli. Herein, we sought to identify and characterize the members of the skeletal muscle secretome, and to determine which protein secretion levels were modulated in response to insulin stimulation. To conduct these studies, we treated differentiated L6 rat skeletal muscle cells with insulin or left them untreated, and we comparatively analyzed the proteins secreted into the media. We fractionated this conditioned media using offline RP HPLC, digested the fractionated proteins, and analyzed the resulting peptides with LC‐ESI‐MS/MS. We identified a total of 254 proteins, and by using three different filtering methods, we identified 153 of these as secretory proteins. Fourteen proteins were secreted at higher levels under insulin stimulation, including several proteins known to be highly secreted in metabolic diseases; 19 proteins were secreted at lower levels under insulin stimulation. These result not only pinpointed several previously unknown, insulin induced, secretory proteins of skeletal muscle, it also described a novel approach for conditioned secretome analysis.     

Cerebrospinal fluid protein patterns in neurodegenerative disease revealed by liquid chromatography-Fourier transform ion cyclotron resonance mass spectrometry

This study demonstrates the power of a novel proteomic approach developed for the detection and identification of biological markers in body fluids. The goal was to observe alterations in the protein patterns of cerebrospinal fluid (CSF) related to amyotrophic lateral sclerosis (ALS), a neurodegenerative disorder with unknown etiology. In the experiments, tryptic digests of CSF from patients and healthy controls were analyzed by on-line capillary liquid chromatography-Fourier transform ion cyclotron resonance mass spectrometry. (FT-ICR MS) Typically, around 4000 peptides were detected in one such experiment, and a pattern recognition program was constructed for the data analysis to distinguish mass chromatograms from patients and controls. This strategy was evaluated comparing the peptide patterns of CSF spiked in vitro with a biomarker, with control CSF. The patterns were clearly separated and the tryptic peptides of the biomarker were successfully selected as characteristic peaks. Hence, the method was applied to compare mass chromatograms of CSF from 12 ALS-patients and 10 matched healthy controls. While no biomarker alone could be identified from the characteristic peaks, we were able to assign 4 out of 5 unknown samples correctly (i.e., 80% correctly diagnosed, 20% false-negative), and it would be 100% if we reject a possible outlier believed to be caused by an occlusion in the spinal CSF compartment. These findings are very promising, although the clinical relevance is not fully established due to the low number of unknown samples analyzed. In addition to the diagnostic potential, these results may be important steps towards understanding the neurodegenerative process.     

Presence of immunoreactive dynorphin in human cerebrospinal fluid

Immunoreactive dynorphin (I-dynorphin) was measured by radioimmunoassay in human cerebrospinal fluid (CSF). I-dynorphin concentration in CSF was 30 +/- 2 pg/ml. Sephadex G-25 column chromatography showed the main peak eluted at the position of dynorphin-(1-17). HPLC elution profile of this major peak from gel filtration showed a large peak corresponding to the position of dynorphin-(1-17) and small peaks corresponding to the positions of dynorphin-(1-13), dynorphin-(1-12) and other unknown peptides.     

Effect of high dose,repeated intra‐cerebrospinal fluid injection of sulphamidase on neuropathology in mucopolysaccharidosis type IIIA mice

Mucopolysaccharidosis type IIIA (MPS IIIA) is an inherited neurodegenerative lysosomal storage disorder characterized by progressive loss of learned skills, sleep disturbance and behavioural problems. Reduced activity of sulphamidase (N‐sulphoglucosamine sulphohydrolase; SGSH; EC 3.10.1.1) results in intracellular accumulation of heparan sulphate (HS), with the brain as the primary site of pathology. We have used a naturally occurring MPS IIIA mouse model to determine the effectiveness of SGSH replacement through the cerebrospinal fluid (CSF) to decrease neuropathology. This is a potential therapeutic option for patients with this disorder. Mice received intra‐CSF injections of recombinant human SGSH (30, 50 or 70 μg) fortnightly from 6 to 18 weeks of age, and the cumulative effect on neuropathology was examined and quantified. Anti‐SGSH antibodies detected in plasma at euthanasia did not appear to impact upon the health of the mice or the experimental outcome, with significant but region‐dependent and dose‐dependent reductions in an HS‐derived oligosaccharide observed in the brain and spinal cord using tandem mass spectrometry. SGSH infusion reduced the number of storage inclusions observed in the brain when visualized using electron microscopy, and this correlated with a significant decrease in the immunohistochemical staining of a lysosomal membrane marker. Reduced numbers of activated isolectin B4‐positive microglia and glial fibrillary acidic protein‐positive astrocytes were seen in many, but not all, brain regions. Significant reductions in the number of ubiquitin‐positive intracellular inclusions were also observed. These outcomes show the effectiveness of this method of enzyme delivery in reducing the spectrum of neuropathological changes in murine MPS IIIA brain.     

电喷雾串联质谱图的叠合与多肽序列分析

利用离子阱电喷雾串联质谱仪,在选择性改变某些食品参数的条件下对模式分子Met-脑啡肽和自行固相化学合成的7肽及其修饰产物、10肽和20肽进行碎裂处理,从而获得一系列具有一定差异的串联质谱图。选择具有适当互补性的图谱进行叠合处理,得到具有连贯性“三联套”（triplet）及“二联套”（doublet）碎片离子峰的叠合串联质谱图,据此可以方便准确地角析出多肽的氨基酸序列。实验结果表明,这种方法在多肽的质谱法测定中具有一定的实用性。     

Synthesis of deoxynivalenol-glucosides and their characterization using a QTrap LC-MS/MS

Deoxynivalenol (DON)-glucosides were successfully synthesized in a two-step reaction from 1-β-Bromo-1-deoxy-2,3,4,6-tetra-O-acetyl-α-D-gluco-pyranose and 3-Acetyl-DON or 15-Acetyl-DON. After purification of the reaction products, the mycotoxin conjugates were for the first time characterized by means of a triple quadrupole mass spectrometer in combination with a linear ion trap. Due to different fragmentation behaviour it was also possible to distinguish between the two glucosides. Presented at the 25^th Mykotoxin Workshop in Giessen, Germany, May 19–21, 2003     

Whole-Cell Protein Identification Using the Concept of Unique Peptides

A concept of unique peptides(CUP)was proposed and implemented to identify whole-cell proteins from tandem mass spectrometry(MS/MS)ion spectra.A unique peptide is defined as a peptide,irrespective of its length,that exists only in one protein of a proteome of interest,despite the fact that this peptide may appear more than once in the same protein.Integrating CUP,a two-step whole-cell protein identification strategy was developed to further increase the confidence of identified proteins.A dataset containing 40,243 MS/MS ion spectra of Saccharomyces cerevisiae and protein identification tools including Mascot and SEQUEST were used to illustrate the proposed concept and strategy.Without implementing CUP,the proteins identified by SEQUEST are 2.26 fold of those identified by Mascot.When CUP was applied,the proteins bearing unique peptides identified by SEQUEST are3.89 fold of those identified by Mascot.By cross-comparing two sets of identified proteins,only 89 common proteins derived from CUP were found.The key discrepancy between identified proteins was resulted from the filtering criteria employed by each protein identification tool.According to the origin of peptides classified by CUP and the commonality of proteins recognized by protein identification tools,all identified proteins were cross-compared,resulting in four groups of proteins possessing different levels of assigned confidence.     

Development and validation of sensitive LC-MS/MS assays for quantification of HP-β-CD in human plasma and CSF

2-Hydroxypropyl-β-cyclodextrin (HP-β-CD), a widely used excipient for drug formulation, has emerged as an investigational new drug for the treatment of Niemann-Pick type C1 (NPC1) disease, a neurodegenerative cholesterol storage disorder. Development of a sensitive quantitative LC-MS/MS assay to monitor the pharmacokinetics (PKs) of HP-β-CD required for clinical trials has been challenging owing to the dispersity of the HP-β-CD. To support a phase 1 clinical trial for ICV delivery of HP-β-CD in NPC1 patients, novel methods for quantification of HP-β-CD in human plasma and cerebrospinal fluid (CSF) using LC-MS/MS were developed and validated: a 2D-LC-in-source fragmentation-MS/MS (2D-LC-IF-MS/MS) assay and a reversed phase ultra performance LC-MS/MS (RP-UPLC-MS/MS) assay. In both assays, protein precipitation and “dilute and shoot” procedures were used to process plasma and CSF, respectively. The assays were fully validated and in close agreement, and allowed determination of PK parameters for HP-β-CD. The LC-MS/MS methods are ∼100-fold more sensitive than the current HPLC assay, and were successfully employed to analyze HP-β-CD in human plasma and CSF samples to support the phase 1 clinical trial of HP-β-CD in NPC1 patients.     

Mass spectrometric analysis of the kinetics of in vivo rhodopsin phosphorylation

On stimulation, rhodopsin, the light-sensing protein in the rod cells of the retina, is phosphorylated at several sites on its C terminus as the first step in deactivation. We have developed a mass spectrometry-based method to quantify the kinetics of phosphorylation at each site in vivo. After exposing either a freshly dissected mouse retina or the eye of an anesthetized mouse to a flash of light, phosphorylation and dephosphorylation reactions are terminated by rapidly homogenizing the retina or enucleated eye in 8 M urea. The C-terminal peptide containing all known phosphorylation sites is cleaved from rhodopsin, partially purified by ultracentrifugation, and analyzed by liquid chromatography coupled with mass spectrometry (LCMS). The mass spectrometer responds linearly to the peptide from 10 fmole to 100 pmole. The relative sensitivity to peptides with zero to five phosphates was determined using purified phosphopeptide standards. High pressure liquid chromatography (HPLC) coupled with tandem mass spectrometry (LCMS/MS) was used to distinguish the three primary sites of phosphorylation, Ser 334, Ser 338, and Ser 343. Peptides monophosphorylated on Ser 334 were separable from those monophosphorylated on Ser 338 and Ser 343 by reversed-phase HPLC. Although peptides monophosphorylated at Ser 338 and Ser 343 normally coelute, the relative amounts of each species in the single peak could be determined by monitoring the ratio of specific daughter ions characteristic of each peptide. Doubly phosphorylated rhodopsin peptides with different sites of phosphorylation also were distinguished by LCMS/MS. The sensitivity of these methods was evaluated by using them to measure rhodopsin phosphorylation stimulated either by light flashes or by continuous illumination over a range of intensities.     

Quantification of Alzheimer amyloid beta peptides ending at residues 40 and 42 by novel ELISA systems

BACKGROUND: The amyloid beta (Abeta) peptide is a key molecule in the pathogenesis of Alzheimer's disease. Reliable methods to detect and quantify soluble forms of this peptide in human biological fluids and in model systems, such as cell cultures and transgenic animals, are of great importance for further understanding the disease mechanisms. In this study, the application of new and highly specific ELISA systems for quantification of Abeta40 and Abeta42 (Abeta peptides ending at residues 40 or 42, respectively) in human cerebrospinal fluid (CSF) are presented. MATERIALS AND METHODS: Monoclonal antibodies WO-2, G2-10 and G2-11 were thoroughly characterized by (SPOT) epitope mapping and immunoprecipitation/mass spectrometry. We determined whether aggregation affected the binding capacities of the antibodies to synthetic peptides and whether components of the CSF affected the ability of the antibodies to bind synthetic Abeta1-40 and Abeta1-42 peptides. The stability of Abeta40 and Abeta42 in CSF during different temperature conditions was also studied to optimize sample handling from lumbar puncture to Abeta assay. RESULTS: The detection range for the ELISAs were 20-250 pM. The intra-assay variations were 2% and 3%, and the inter-assay variations were 2% and 10% for Abeta40 and Abeta42, respectively. The antibodies specifically detected the expected peptides with equal affinity for soluble and fibrillar forms of the peptide. The presence of CSF obstructed the recognition of synthetic peptides by the antibodies and the immunoreactivity of endogenous CSF Abeta decreased with increasing storage time and temperature. CONCLUSIONS: This study describes highly sensitive ELISAs with thoroughly characterized antibodies for quantification of Abeta40 and Abeta42, an important tool for the understanding of the pathogenesis of Alzheimer's disease. Our results pinpoint some of the difficulties associated with Abeta quantification and emphasize the importance of using a well-documented assay.     

Pro-enkephalin peptides possessing Met-enkephalin-Arg6-Gly7-Leu8-immunoreactivity in rat CSF, striatum and adrenal gland

Cerebrospinal fluid (CSF) taken from rats implanted with chronic cisternal cannulae and extracts prepared from rat adrenal gland and striatum were subjected to Sephadex G-50 chromatography and HPLC. Fractions were monitored using specific radioimmunoassays (RIA) for the pentapeptide methionine enkephalin (Met-Enk) and methionine enkephalin-Arg6-Gly7-Leu8 (Met-EnkRGL). In rat CSF, striatum and adrenal gland, three Met-EnkRGL-immunoreactive (IR) peaks of Mrs 8000, 5000 and 1000 daltons were detected. The same peaks were also found to possess Met-Enk-immunoreactivity after enzyme digestion of Sephadex G-50 fractions with trypsin and carboxypeptidase B (CPB), suggesting their derivation from proenkephalin. HPLC of the 8K and 5K peaks on a column of Ultrapore RPSC showed them to elute discretely with similar retention times, indicative of hydrophobic peptides of large molecular weight. Their similar hydrophobicities yet significant separation during gel filtration would suggest that the 8K and 5K peptides are structurally closely related yet different with respect to their molecular weights. HPLC of the small molecular weight material from rat striatum and adrenal gland revealed the presence of Met-EnkRGL and Met-EnkRGL sulphoxide in both tissues. In rat striatum Met-Enk and its sulphoxide were also detected. The oxidised pentapeptide was found to be present in rat CSF, together with two unidentified small molecular weight Met-Enk-IR peaks detected without prior enzyme digestion of fractions. The small molecular weight Met-EnkRGL-IR material in rat CSF was found to be comprised of two unknown peptides which were less hydrophobic than Met-EnkRGL and its sulphoxide derivative.