Improvement of an in-gel tryptic digestion method for matrix-assisted laser desorption/ionization-time of flight mass spectrometry peptide mapping by use of volatile solubilizing agents

The combination of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), in-gel enzymatic digestion of proteins separated by two-dimensional gel electrophoresis and searches of molecular weight in peptide-mass databases is a powerful and well established method for protein identification in proteomics analysis. For successful protein identification by MALDI-TOF mass spectrometry of peptide mixtures, critical parameters include highly specific enzymatic cleavage, high mass accuracy and sufficient numbers and sequence coverage of the peptides which can be analyzed. For in-gel digestion with trypsin, the method employed should be compatible both with enzymatic cleavage and subsequent MALDI-TOF MS analysis. We report here an improved method for preparation of peptides for MALDI-TOF MS mass fingerprinting by using volatile solubilizing agents during the in-gel digestion procedure. Our study clearly demonstrates that modification of the in-gel digestion protocols by addition of dimethyl formamide (DMF) or a mixture of DMF/N,N-dimethyl acetamide at various concentrations can significantly increase the recovery of peptides. These higher yields of peptides resulted in more effective protein identification.     

Affinity-tagged phosphorylation assay by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (ATPA-MALDI): application to calcium/calmodulin-dependent protein kinase

A matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS)-based kinase assay using a peptide substrate tagged with a biotinyl group has been developed. The peptide moiety was designed to serve as an efficient substrate for calcium/calmodulin-dependent protein kinase II, based on the in vivo phosphorylation site of phosrestin I, a Drosophila homolog of arrestin. In the assay, the quantitative relationship was determined from the ratio of the peak areas between the two peaks respectively representing the unphosphorylated and the phosphorylated substrate. Attempts to assay phosphorylated peptides directly from the reaction mixture, gave inaccurate results because of the high noise level caused by the presence of salts and detergents. In contrast, after purifying the substrate peptides with the biotin affinity tag using streptavidin-coated magnetic beads, peak areas accurately represented the ratio between the unphosphorylated and phosphorylated peptide. By changing the substrate peptide to a peptide sequence that serves as a kinase substrate, it is expected that an efficient non-radioactive protein kinase assay using MALDI-TOF MS can be developed for any type of protein kinase. We call this technique "Affinity-Tagged Phosphorylation Assay by MALDI-TOF MS (ATPA-MALDI)." ATPA-MALDI should serve as a quick and efficient non-radioactive protein kinase assay by MALDI-TOF MS.     

Increased sensitivity of tryptic peptide detection by MALDI-TOF mass spectrometry is achieved by conversion of lysine to homoarginine

Mass spectrometric techniques for identification of proteins by "mass fingerprinting" (matching the masses of tryptic peptides from a protein digest to the theoretical peptides in a database) such as matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) are rapidly growing in popularity as the demand for high throughput analysis of the proteome increases. This is due, in part, to the ability to automate the technique and the rapid rate with which mass spectra may be acquired. An important factor in the accuracy of the technique is the number of tryptic peptides that are identified in the various searching algorithms that exist. The greater sequence coverage of the parent protein that is obtained, the higher the level of confidence in the identification that is determined. One impediment to high levels of sequence coverage is the bias of MALDI-TOF mass spectrometry to arginine-containing peptides. Increasing the sensitivity to lysine-containing peptides should increase the sequence coverage obtained. In order to achieve this result we have developed conditions to modify the epsilon-amine group of lysine in tryptic peptides with O-methylisourea. The conditions utilized result in the conversion of lysine to homoarginine with no modification of the amine terminus of the peptides. The sensitivity of MALDI-TOF mass spectrometry detection of peptides was increased dramatically following modification. The modification chemistry may be applied to tryptic peptide mixtures prior to desalting and spotting onto MALDI-TOF plates. This technique will be particularly useful for identifying proteins with a high lysine/arginine ratio.     

Citraconylation--a simple method for high protein sequence coverage in MALDI-TOF mass spectrometry

Lysine epsilon -amino group reacts with citraconic anhydride forming a derivative, which is stable on terms for trypsin cleavage. This modification changes the spectrum of peptides formed by the trypsin action; as the number of trypsin-sensitive sites is reduced, the peptides with higher molecular mass can survive in the digest. The various studies of proteins by MALDI-TOF mass spectrometry are often complicated by the low sequence coverage of the peptide chain. This paper demonstrates that the modification of proteins by citraconylation before trypsin cleavage represents a simple experimental technique, which allows a significant increase of sequence coverage in MALDI-TOF mass spectrometry. This improvement is caused both by change of trypsin fragmentation pattern and by disturbance of the protein's native tertiary structure.     

Using proteomics to mine genome sequences

We present a method for mining unannotated or annotated genome sequences with proteomic data to identify open reading frames. The region of a genome coding for a protein sequence is identified by using information from the analysis of proteins and peptides with MALDI-TOF mass spectrometry. The raw genome sequence or any unassembled contigs of an organism are theoretically cleaved into a number of equal sized but overlapping fragments, and these are then translated in all six frames into a series of virtual proteins. Each virtual protein is then subjected to a theoretical enzymatic digestion. Standard proteomic sample preparation methods are used to separate, array, and digest the proteins of interest to peptides. The masses of the resulting peptides are measured using mass spectrometry and compared to the theoretical peptide masses of the virtual proteins. The region of the genome responsible for coding for a particular protein can then be identified when there are a large number of hits between peptides from the protein and peptides from the virtual protein. The method makes no assumptions about the location of a protein in a particular gene sequence or the positions or types of start and stop codons. To illustrate this approach, all 773 proteins of Pseudomonas aeruginosa contained in SWISS-PROT were used to theoretically test the method and optimize parameters. Increasing the size of the virtual proteins results in an overall improvement in the ability to detect the coding region, at the cost of decreasing the sensitivity of the method for smaller proteins. Increasing the minimum number of matching peptides, lowering the mass error tolerance, or increasing the signal-to-noise ratio of the simulated mass spectrum, improves the ability to detect coding regions. The method is further demonstrated on experimental data from Mycobacterium tuberculosis and is also shown to work with eukaryotic organisms (e.g., Homo sapiens).     

De novo mass spectrometry sequencing and characterization of species-specific peptides from nucleoside diphosphate kinase B for the classification of commercial fish species belonging to the family Merlucciidae

The characterization by de novo peptide sequencing of the different protein nucleoside diphosphate kinase B (NDK B) from all the commercial hakes and grenadiers belonging to the family Merlucciidae is reported. A classical proteomics approach, consisting of two-dimmensional gel electrophoresis, tryptic in-gel digestion of the excised spots, MALDI-TOF MS, LC-MS/MS, and nanoESI-MS/MS analyses, was followed for the purification and characterization of the different isoforms of the NDK B. Fragmentation spectra were used for de novo peptide sequence. A high degree of homology was found between the sequences of all the species studied and the NDK B sequence from Gillichthys mirabilis, which is accessible in the protein databases. Particular attention was paid to the differential characterization of species-specific peptides that could be used for fish authentication purposes. These findings allowed us to propose a rapid and effective classification method, based in the detection of these biomarker peptides using the selective ion reaction monitoring (SIRM) scan mode in mass spectrometry.     

A direct approach to quantification of the cellular uptake of cell-penetrating peptides using MALDI-TOF mass spectrometry

This protocol allows the accurate quantification of cell-penetrating peptide (CPP) cellular uptake by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). Quantification is based on the use of an internal standard with same chemical structure as the analyte but labeled with a stable isotope. The analyte and the standard can both be obtained by standard solid-phase peptide synthesis using commercially available amino acids. They are functionalized by biotin to allow their easy purification before MALDI-TOF MS analysis. The method allows determination of the amount of intact internalized peptide and the identification of potential intracellular digests. It can be used to simultaneously compare the uptake of several peptides, and can also be applied to the quantification of peptidic cargoes and the study of their intracellular stability. It is therefore a potent tool to study the mechanisms of CPPs internalization and to select new carriers for drug delivery. This protocol will take approximately 5 hours for the analysis of 12 samples (not including the time for cell incubation with peptides).     

Matrix-assisted laser desorption/ionization-tandem mass spectrometry with high resolution and sensitivity for identification and characterization of proteins

Although peptide mass fingerprinting is currently the method of choice to identify proteins, the number of proteins available in databases is increasing constantly, and hence, the advantage of having sequence data on a selected peptide, in order to increase the effectiveness of database searching, is more crucial. Until recently, the ability to identify proteins based on the peptide sequence was essentially limited to the use of electrospray ionization tandem mass spectrometry (MS) methods. The recent development of new instruments with matrix-assisted laser desorption/ionization (MALDI) sources and true tandem mass spectrometry (MS/MS) capabilities creates the capacity to obtain high quality tandem mass spectra of peptides. In this work, using the new high resolution tandem time of flight MALDI-(TOF/TOF) mass spectrometer from Applied Biosystems, examples of successful identification and characterization of bovine heart proteins (SWISS-PROT entries: P02192, Q9XSC6, P13620) separated by two-dimensional electrophoresis and blotted onto polyvinylidene difluoride membrane are described. Tryptic protein digests were analyzed by MALDI-TOF to identify peptide masses afterward used for MS/MS. Subsequent high energy MALDI-TOF/TOF collision-induced dissociation spectra were recorded on selected ions. All data, both MS and MS/MS, were recorded on the same instrument. Tandem mass spectra were submitted to database searching using MS-Tag or were manually de novo sequenced. An interesting modification of a tryptophan residue, a "double oxidation", came to light during these analyses.     

A new chemical approach to differentiate carboxy terminal peptide fragments in cyanogen bromide digests of proteins

We present a novel approach to perform C-terminal sequence analysis by discriminating the C-terminal peptide in a mass spectral analysis of a CNBr digest. During CNBr cleavage, all Met-Xxx peptide bonds are cleaved and the generated internal peptides all end with a homoserine lactone (hsl)-derivative. The partial opening of the hsl-derivatives, by using a slightly basic buffer solution, results in the formation of m/z doublets (Δm = 18 Da) for all internal peptides and allows to identify the C-terminal peptide which appears as a singlet in the mass spectra. Using two model proteins we demonstrate that this approach can be applied to study proteins purified in gel or in solution. The chemical opening of the hsl-derivative does not require any sample clean-up and therefore, the sensitivity of the C-terminal sequencing approach is increased significantly. Finally, the new protocol was applied to characterize the C-terminal sequence of two recombinant proteins. Tandem mass spectrometry by MALDI-TOF/TOF allowed to identify the sequence of the C-terminal peptides. This novel approach will allow to perform a proteome-wide study of C-terminal proteolytic processing events in a high-throughput fashion.     

Neuropeptidomic analysis of the brain and thoracic ganglion from the Jonah crab,Cancer borealis

Mass spectrometric methods were applied to determine the peptidome of the brain and thoracic ganglion of the Jonah crab (Cancer borealis). Fractions obtained by high performance liquid chromatography were characterized using MALDI-TOF MS and ESI-Q-TOF MS/MS. In total, 28 peptides were identified within the molecular mass range 750-3000Da. Comparison of the molecular masses obtained with MALDI-TOF MS with the calculated molecular masses of known crustacean peptides revealed the presence of at least nine allatostatins, three orcokinin precursor derived peptides, namely FDAFTTGFGHS, [Ala(13)]-orcokinin, and [Val(13)]-orcokinin, and two kinins, a tachykinin-related peptide and four FMRFamide-related peptides. Eight other peptides were de novo sequenced by collision induced dissociation on the Q-TOF system and yielded AYNRSFLRFamide, PELDHVFLRFamide or EPLDHVFLRFamide, APQRNFLRFamide, LNPFLRFamide, DVRTPALRLRFamide, and LRNLRFamide, which belong to the FMRFamide related peptide family, as well as NFDEIDRSGFA and NFDEIDRSSFGFV, which display high sequence similarity to peptide sequences within the orcokinin precursor of Orconectes limosus. Our paper is the first (neuro)peptidomic analysis of the crustacean nervous system.     

Membrane binding properties and terminal residues of the mature hepatitis C virus capsid protein in insect cells

The immature core protein (p23, residues 1 to 191) of hepatitis C virus undergoes posttranslational modifications including intramembranous proteolysis within its C-terminal signal sequence by signal peptide peptidase to generate the mature form (p21). In this study, we analyzed the cleavage site and other amino acid modifications that occur on the core protein. To produce the posttranslationally modified core protein, we used a baculovirus-insect cell expression model system. As previously reported, p23 is processed to form p21 in insect as well as in mammalian cells. p21 was found to be associated with the cytoplasmic membrane, and its significant portion behaved as an integral membrane protein. The protein was purified from the membrane by a simple and unique procedure on the basis of its membrane-binding properties and solubility in detergents. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis of purified p21 showed that the average molecular mass (m/z 19,307) of its single-charged ion differs by m/z 1,457 from that calculated for p23. To determine the posttranslational modifications, tryptic p21 peptides were analyzed by MALDI-TOF MS. We found three peptides that did not match the theoretically derived peptides of p23. Analysis of these peptides by MALDI-TOF tandem MS revealed that they correspond to N-terminal peptides (residues 2 to 9 and 2 to 10) starting with alpha-N-acetylserine and C-terminal peptide (residues 150 to 177) ending with phenylalanine. These results suggest that the mature core protein (molecular mass of 19,306 Da) includes residues 2 to 177 and that its N terminus is blocked with an acetyl group.     

Precision of heavy-light peptide ratios measured by maldi-tof mass spectrometry

We have investigated the precision of peptide quantitation by MALDI-TOF mass spectrometry (MS) using six pairs of proteotypic peptides (light) and same-sequence stable isotope labeled synthetic internal standards (heavy). These were combined in two types of dilution curves spanning 100-fold and 2000-fold ratios. Coefficients of variation (CV; standard deviation divided by mean value) were examined across replicate MALDI spots using a reflector acquisition method requiring 100?000 counts for the most intense peak in each summed spectrum. The CV of light/heavy peptide centroid peak area ratios determined on four replicate spots per sample, averaged across 11 points of a 100-fold dilution curve and over all six peptides, was 2.2% (ranging from 1.5 to 3.7% among peptides) at 55 fmol total (light + heavy) of each peptide applied per spot, and 2.5% at 11 fmol applied. The average CV of measurements at near-equivalence (light = heavy, the center of the dilution curve) for the six peptides was 1.0%, about 17-fold lower CV than that observed when five peptides were ratioed to a sixth peptide (i.e., a different-sequence internal standard). Response curves across the 100-fold range were not completely linear but could be closely modeled by a power law fit giving R(2) values >0.998 for all peptides. The MALDI-TOF MS method was used to determine the endogenous level of a proteotypic peptide (EDQYHYLLDR) of human protein C inhibitor (PCI) in a plasma digest after enrichment by capture on a high affinity antipeptide antibody, a technique called stable isotope standards and capture by anti-peptide antibodies (SISCAPA). The level of PCI was determined to be 770 ng/mL with a replicate measurement CV of 1.5% and a >14?000-fold target enrichment via SISCAPA-MALDI-TOF. These results indicate that MALDI-TOF technology can provide precise quantitation of high-to-medium abundance peptide biomarkers over a 100-fold dynamic range when ratioed to same-sequence labeled internal standards and enriched to near purity by specific antibody capture. The robustness and throughput of MALDI-TOF in comparison to conventional nano-LC-MS technology could enable currently impractical large-scale verification studies of protein biomarkers.     

Salivary agglutinin, which binds Streptococcus mutans and Helicobacter pylori, is the lung scavenger receptor cysteine-rich protein gp-340

Salivary agglutinin is a high molecular mass component of human saliva that binds Streptococcus mutans, an oral bacterium implicated in dental caries. To study its protein sequence, we isolated the agglutinin from human parotid saliva. After trypsin digestion, a portion was analyzed by matrix-assisted laser/desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), which gave the molecular mass of 14 unique peptides. The remainder of the digest was subjected to high performance liquid chromatography, and the separated peptides were analyzed by MALDI-TOF/post-source decay; the spectra gave the sequences of five peptides. The molecular mass and peptide sequence information showed that salivary agglutinin peptides were identical to sequences in lung (lavage) gp-340, a member of the scavenger receptor cysteine-rich protein family. Immunoblotting with antibodies that specifically recognized either lung gp-340 or the agglutinin confirmed that the salivary agglutinin was gp-340. Immunoblotting with an antibody specific to the sialyl Le(x) carbohydrate epitope detected expression on the salivary but not the lung glycoprotein, possible evidence of different glycoforms. The salivary agglutinin also interacted with Helicobacter pylori, implicated in gastritis and peptic ulcer disease, Streptococcus agalactiae, implicated in neonatal meningitis, and several oral commensal streptococci. These results identify the salivary agglutinin as gp-340 and suggest it binds bacteria that are important determinants of either the oral ecology or systemic diseases.     

Immunocytochemical and molecular data guide peptide identification by mass spectrometry: orcokinin and orcomyotropin-related peptides in the stomatogastric nervous system of several crustacean species.

In order to identify new orcokinin and orcomyotropin-related peptides in crustaceans, molecular and immunocytochemical data were combined with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). In the crayfish Procambarus clarkii, four orcokinins and an orcomyotropin-related peptide are present on the precursor. Because these peptides are highly conserved, we assumed that other species have an identical number of peptides. To identify the peptides, immunocytochemistry was used to localize the regions of the stomatogastric nervous system in which orcokinins are predominantly present. One of the regions predominantly containing orcokinins was a previously undescribed olive-shaped neuropil region within the commissural ganglia of the lobsters Homarus americanus and Homarus gammarus. MALDI-TOF MS on these regions identified peptide masses that always occur together with the known orcokinins. Seven peptide ions occurred together in the peptide massspectra of the lobsters. Mass spectrometric fragmentation by MALDI-MS post-source decay (PSD) and electrospray ionization quadrupole time-of-flight mass spectrometry (ESI Q-TOF MS) collision-induced dissociation (CID) were used in the identification of six of these masses, either as orcokinins or as orcomyotropin-related peptides and revealed three hitherto unknown peptide variants, two of which are [His13]-orcokinin ([M+H]+ = 1540.8 Da) and an orcomyotropin-related peptide FDAFTTGFGHN ([M+H]+ = 1213.5 Da). The mass of the third previously unknown orcokinin variant corresponded to that of an identified orcokinin, but PSD fragmentation did not support the suggested amino acid sequence. CID analysis allowed partial de novo sequencing of this peptide. In the crab Cancer pagurus, five orcokinins and an orcomyotropin-related peptide were unambigously identified, including the previously unknown peptide variant [Ser9-Val13]-orcokinin ([M+H]+ = 1532.8 Da).     

Analysis of human serum proteins by liquid phase isoelectric focusing and matrix-assisted laser desorption/ionization-mass spectrometry

Direct matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis of human serum yielded ion signals from only a fraction of the total number of peptides and proteins expected to be in the sample. We increased the number of peptide and protein ion signals observed in the MALDI-TOF mass spectra analysis of human serum by using a prefractionation protocol based on liquid phase isoelectric focusing electrophoresis. This pre-fractionation technique facilitated the MALDI-TOF MS detection of as many as 262 different peptide and protein ion signals from human serum. The results obtained from three replicate fractionation experiments on the same serum sample indicated that 148 different peptide and protein ion signals were reproducibly detected using our isoelectric focusing and MALDI-TOF MS protocol.     

A comparison of the neuropeptides from the retrocerebral complex of adult male and female Manduca sexta using MALDI-TOF mass spectrometry

The occurrence of neuropeptides in the retrocerebral complexes of adult male and females of the tobacco hawkmoth, Manduca sexta, was investigated using matrix-assisted laser desorption time of flight (MALDI-TOF) mass spectrometry (MS), post source decay (PSD) and collision-induced dissociation (CID) MS/MS. From fractions of methanol extracts of corpora cardiaca (CC)/corpora allata (CA), separated by reversed-phase high performance liquid chromatography (RP-HPLC), a total of 11 mass ions were assigned to known peptides from M. sexta. These peptides were adipokinetic hormone (AKH), FLRFamides I, II and III, crustacean cardioactive peptide (CCAP), cardioactive peptide 2b (CAP(2b)), three myoinhibitory peptides, corazonin, and M. sexta allatostatin (Manse-AS). A further six masses were in agreement with Y/FXFGLamide allatostatins identified from other Lepidoptera. The sequence identities of FLRFamide I and AKH were confirmed using post source decay analysis. Fragmentation by collision-induced dissociation MS/MS identified an extended AKH peptide. The apparent differences in the peptides present in male and female retrocerebral complexes are most likely quantitative rather than sex specific.     

Technical evaluation of MALDI-TOF mass spectrometry for quantitative proteomic profiling matrix formulation and application

Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) has been recently used to identify disease markers by directly profiling and quantifying the peptide/proteins in biological samples under different physiological or experimental conditions. The information of reproducibility of such quantitative profiling method has not been available. It is important to evaluate and reduce error from technical variation. In this study, an unbiased signal acquisition strategy was used to evaluate the effects of three sample-matrix spotting methods and two matrix chemicals, α-cyano-4-hydroxycinnamic acid (CHCA) and sinapinic acid, on the reproducibility of the peptide/protein signal intensities. The sandwich spotting method using 0.1% nitrocellulose coating film and CHCA gave the best quantitative results for the standard peptides and proteins with mass<66.5 kDa. The normalized signal intensities of the standard peptides and proteins were directly proportional to their concentrations with intra-assay (within-day) coefficient of variations (CVs) ranging from 6.5% to 17%. When analyzing serum peptides <6000 m/z, the interassay (between-days) CVs of all the evaluated peptide peaks were <15%. These data indicate that with the right MS analysis conditions, MALDI-TOF MS appears to be a feasible tool for directly profiling and quantifying the peptide/ proteins in biological samples.     

Simple dual-spotting procedure enhances nLC-MALDI MS/MS analysis of digests with less specific enzymes

The beneficial effect of high mass accuracy in mass spectrometry is especially pronounced when using less specific enzymes as the number of theoretically possible peptides increases dramatically without any cleavage specificity defined. Together with a preceding chromatographic separation, high-resolution mass spectrometers such as the MALDI-LTQ-Orbitrap are therefore well suited for the analysis of protein digests with less specific enzymes. A combination with fast, automated, and informative MALDI-TOF/TOF analysis has already been shown to yield increased total peptide and protein identifications. Here, a simple method for nLC separation and subsequent alternating spotting on two targets for both a MALDI-LTQ-Orbitrap and a MALDI-TOF/TOF instrument is introduced. This allows for simultaneous measurements on both instruments and subsequent combination of both data sets by an in-house written software tool. The performance of this procedure was evaluated using a mixture of four standard proteins digested with elastase. Three replicate runs were examined concerning repeatability and the total information received from both instruments. A cytosolic extract of C. glutamicum was used to demonstrate the applicability to more complex samples. Database search results showed that an additional 32.3% of identified peptides were found using combined data sets in comparison to MALDI-TOF/TOF data sets.     

Mass spectrometric analysis of single identified neurons of an insect

The combination of retrograde labelling with dextran-tetramethylrhodamine and MALDI-TOF mass spectrometry was used to analyse for the first time the peptidome of a series of morphologically identified single neurosecretory cells of an insect. Eight postero-lateral cells of the metathoracic ganglion of the American cockroach, Periplaneta americana, were used to demonstrate that: (1) the complete dissection procedure can be documented and (2) the mass spectrometric analysis of the dissected somata results in highly reproducible mass spectra. In total, 21 FMRFamide-related peptides were detected in each of the postero-lateral cells which release their neurosecretions via thoracic perisympathetic organs. Direct analysis of these neurohemal organs confirmed the co-storage of FMRFamide-related peptides. Two additional abundant peptides from thoracic perisympathetic organs which were not detectable in the postero-lateral cells were characterized using ESI-Q-TOF MS/MS. De novo sequencing yielded two related peptides (FERL/IEamides) without any similarity with known peptide families of insects.     

Fertilization in Sepia officinalis: the first mollusk sperm-attracting peptide

Egg mass extract was used to characterize regulatory peptides, involved in the successive steps of egg-laying of the cuttlefish Sepia officinalis. Among these peptides, a C-terminally amidated hexapeptide revealed a sperm-attracting activity. MALDI-TOF MS (matrix-assisted laser desorption ionization-time of flight mass spectrometry) and Edman degradation led to a peptide of m/z 596.6 and the following primary sequence: Pro-Ile-Asp-Pro-Gly-Val-CO(NH2). From concentrations as low as 10(-17)M, the PIDPGVamide was able to attract freshly dissected spermatozoa. Nano-ESI-Q-TOF MS (nano-electrospray ionization-quadrupole-time-of-flight mass spectrometry) analysis established the quantitative occurrence of this peptide in different egg structures. The PIDPGVamide appears to be synthesized in oocytes during vitellogenesis and released by the embedded oocytes in the external media during egg-laying to facilitate fertilization by increasing chances of gamete collision. This novel peptide called SepSAP for Sepia sperm attracting peptide is the first sperm-attracting peptide, identified in mollusks or even in protostomians.