Multifunctional ZrO2 nanoparticles and ZrO2-SiO2 nanorods for improved MALDI-MS analysis of cyclodextrins, peptides, and phosphoproteins

Multifunctional ZrO₂ nanoparticles (NPs) and ZrO₂-SiO₂ nanorods (NRs) have been successfully applied as the matrices for cyclodextrins and as affinity probes for enrichment of peptides (leucine-enkephalin, methionine-enkephalin and thiopeptide), phosphopeptides (from tryptic digestion products of β-casein) and phosphoproteins from complex samples (urine and milk) in atmospheric pressure matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) and MALDI time-of-flight (TOF) MS. The results show that the ZrO₂ NPs and ZrO₂-SiO₂ NRs can interact with target molecules (cyclodextrins, peptides, and proteins), and the signal intensities of the analytes were significantly improved in MALDI-MS. The maximum signal intensities of the peptides were obtained at pH 4.5 using ZrO₂ NPs and ZrO₂-SiO₂ NRs as affinity probes. The limits of detection of the peptides were found to be 75-105 fmol for atmospheric pressure MALDI-MS and those of the cyclodextrins and β-casein were found to be 7.5-20 and 115-125 fmol, respectively, for MALDI-TOF-MS. In addition, these nanomaterials can be applied as the matrices for the analysis of cyclodextrins in urine samples by MALDI-TOF-MS. ZrO₂ NPs and ZrO₂-SiO₂ NRs efficiently served as electrostatic probes for peptide mixtures and milk proteins because 2–11 times signal enhancement can be achieved compared with use of conventional organic matrices. Moreover, we have successfully demonstrated that the ZrO₂ NPs can be effectively applied for enrichment of phosphopeptides from tryptic digestion of β-casein. Comparing ZrO₂ NPs with ZrO₂-SiO₂ NRs, we found that ZrO₂ NPs exhibited better affinity towards phosphopeptides than ZrO₂-SiO₂ NRs. Furthermore, the ZrO₂ and ZrO₂-SiO₂ nanomaterials could be used to concentrate trace amounts of peptides/proteins from aqueous solutions without tedious washing procedures. This approach is a simple, straightforward, separation-and washing-free approach for MALDI-MS analysis of cyclodextrins, peptides, proteins, and tryptic digestion products of phosphoproteins.      

Molecular aspects of milk allergens and their role in clinical events

Milk allergy is the most frequent food allergy in childhood. Even though cases of newly developed milk allergy in adulthood are known, this allergy is less frequent in adults since it is normally outgrown by children during the first years of life. One of the reasons why allergy to cow’s milk shows its highest prevalence in children is its early introduction into the diets of babies when breast feeding is not possible. The major allergens are caseins and β-lactoglobulin, but allergies to other minor proteins (immunoglobulins, bovine serum albumin) have also been reported. Milk allergenicity can be reduced by various treatments (mainly hydrolysis), meaning that formulas based on cow’s milk can often be safely fed to children allergic to milk proteins. Cross-reactivity has been described between different mammalian milks and between milk and meat or animal dander. Cross-contamination can result from inadequate cleaning of industrial equipment and constitutes a hidden danger for allergic subjects who unknowingly ingest milk proteins. Figure Involvement (expressed as percentage of total subjects) of the most abundant milk proteins in the sensitization of 80 children allergic to cow’s milk. The upper panel includes all positive responses, even minor ones; data in the lower panel are restricted to the most severe positive responses (see text for details). SPT, skin prick test; CAP, CAP test; IMM, immunoblotting; alpha-LA, α-lactalbumin; beta-LG, β-lactoglobulin; cas, caseins; BSA, bovine serum albumin     

Increasing charge while preserving noncovalent protein complexes for ESI-MS

Increased multiple charging of native proteins and noncovalent protein complexes is observed in electrospray ionization (ESI) mass spectra obtained from nondenaturing protein solutions containing up to 1% (vol/vol) m-nitrobenzyl alcohol (m-NBA). The increases in charge ranged from 8% for the 690 kDa α₇β₇β₇α₇ 20S proteasome complex to 48% additional charge for the zinc-bound 29 kDa carbonic anhydrase-II protein. No dissociation of the noncovalently bound ligands/subunits was observed upon the addition of m-NBA. It is not clear if the enhanced charging is related to altered surface tension as proposed in the “supercharging” model of Iavarone and Williams (Iavarone, A. T.; Williams, E. R. J. Am. Chem. Soc. 2003, 125, 2319–2327). However, more highly charged noncovalent protein complexes have utility in relaxing slightly the mass-to-charge (m/z) requirements of the mass spectrometer for detection and will be effective for enhancing the efficiency for tandem mass spectrometry studies of protein complexes.     

Charge remote fragmentation in electron capture and electron transfer dissociation

Secondary fragmentations of three synthetic peptides (human αA crystallin peptide 1-11, the deamidated form of human βB2 crystallin peptide 4-14, and amyloid β peptide 25-35) were studied in both electron capture dissociation (ECD) and electron-transfer dissociation (ETD) mode. In ECD, in addition to c and z· ion formations, charge remote fragmentations (CRF) of z· ions were abundant, resulting in internal fragment formation or partial/entire side-chain losses from amino acids, sometimes several residues away from the backbone cleavage site, and to some extent multiple side-chain losses. The internal fragments were observed in peptides with basic residues located in the middle of the sequences, which was different from most tryptic peptides with basic residues located at the C-terminus. These secondary cleavages were initiated by hydrogen abstraction at the α-, β-, or γ-position of the amino acid side chain. In comparison, ETD generates fewer CRF fragments than ECD. This secondary cleavage study will facilitate ECD/ETD spectra interpretation, and help de novo sequencing and database searching.     

Detection of residual bacitracin A, colistin A, and colistin B in milk and animal tissues by liquid chromatography tandem mass spectrometry

Liquid chromatography coupled with electrospray ionization tandem mass spectrometry (LC-MS/MS) was applied to the determination of residual bacitracin A, colistin A, and colistin B in milk and animal tissue samples. Prior to instrumental analysis, samples were subjected to acid extraction followed by solid-phase cleanup using Strata-X cartridges. Mass spectral acquisitions were performed under selective multiple reaction monitoring (MRM) mode at m/z 199 and 670 from triply charged precursors of bacitracin A (m/z 475); m/z 385 and 379 from triply charged precursors of colistin A (m/z 391); and m/z 380 and 374 from triply charged precursors of colistin B (m/z 386). Method precision was evaluated from spike recovery of samples fortified at concentrations corresponding to 2/5 of the maximum residue limits (MRLs) for each of the analytes under study. Intra-day and inter-day variations were found to range from 90.9 to 104% with relative standard deviation (RSD) <6.5%, and from 90.1 to 106% with RSD <9.1%, respectively. Limits of quantification (LOQs) were defined as the spiking concentrations at 2/5 MRL, and limits of detection (LODs) were 10–47 μg kg⁻¹ for bacitracin A, 1–16 μg kg⁻¹ for colistin A, and 6–14 μg kg⁻¹ for colistin B in milk and animal tissues. The presented method has good precision and high sensitivity and was applied as a fast screening protocol and a quantitative tool for monitoring of the concerned polypeptides in foods as part of a surveillance program.      

Lactosylated casein phosphopeptides as specific indicators of heated milks

Casein phosphopeptides (CPP) were identified in small amounts in milks heated at various intensities by using matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry. CPP selectively concentrated on hydroxyapatite (HA) were regenerated using phosphoric acid mixed in the matrix. Unphosphorylated peptides not retained by HA were removed by buffer washing. This procedure enhanced the MALDI signals of CPP that are ordinarily suppressed by the co-occurrence of unphosphorylated peptides. CPP, belonging to the β-casein (CN) family, i.e., (f1-29) 4P, (f1-28) 4P, and (f1-27) 4P, and the α_s2-CN family, i.e., (f1-21) 4P and (f1-24) 4P, were observed in liquid and powder milk. The lactosylated counterparts were specific to intensely heated milks, but absent in raw and thermized/pasteurized milk. Most CPP with C-terminal lysines probably arose from the activity of plasmin; an enzyme most active in casein hydrolysis. A CPP analogue was used as the internal standard. The raw milk signature peptide β-CN (f1-28) 4P constituted ~4.3% of the total β-CN. Small amounts of lactosylated peptides, which varied with heat treatment intensity, were detected in the milk samples. The limit of detection of ultra-high-temperature milk adjunction in raw or pasteurized milk was ~10%.     

MS–MS Fragmentation Patterns of Cholesterol Oxidation Products

Summary. This study was aimed at analyzing the daughter ion spectra of 7 toxicologically relevant cholesterol oxidation products (COPs) i.e. cholestanetriol (cholestane-3β,5α,6β-triol), 7-ketocholesterol (cholest-5-en-3β-ol-7-one), 7α-hydroxycholesterol (cholest-5-en-3β,7α-diol), 7β-hydroxycholesterol (cholest-5-en-3β,7β-diol), 25-hydroxycholesterol (cholest-5-en-3β,25-diol), α-epoxycholesterol (cholestane-5α,6α-epoxy-3β-ol) and β-epoxycholesterol (cholestane-5β,6β-epoxy-3β-ol). In addition, 19-hydroxycholesterol (cholest-5-en-3β,19-diol) was analyzed as this serves as internal standard in COPs determination by HPLC-MS. Mass spectrometry was performed using a triple quadrupol mass spectrometer that was equipped with an APCI ion source. Our results indicate a common fragmentation pattern for COPs. The main breaking sites identified were in the sterol ring system between the carbon atoms with the position numbers 11–12, 12–13, and 8–14. Typical daughter ions of m/z = 81, 95, and 195 were used for multiple reaction monitoring analysis.     

Unusual Fragmentation of β-Linked Peptides by ExD Tandem Mass Spectrometry

Ion-electron reaction based fragmentation methods (ExD) in tandem mass spectrometry (MS), such as electron capture dissociation (ECD) and electron transfer dissociation (ETD) represent a powerful tool for biological analysis. ExD methods have been used to differentiate the presence of the isoaspartate (isoAsp) from the aspartate (Asp) in peptides and proteins. IsoAsp is a β₃-type amino acid that has an additional methylene group in the backbone, forming a C_α–C_β bond within the polypeptide chain. Cleavage of this bond provides specific fragments that allow differentiation of the isomers. The presence of a C_α–C_β bond within the backbone is unique to β-amino acids, suggesting a similar application of ExD toward the analysis of peptides containing other β-type amino acids. In the current study, ECD and ETD analysis of several β-amino acid containing peptides was performed. It was found that N–C_β and C_α–C_β bond cleavages were rare, providing few c and z type fragments, which was attributed to the instability of the C_β radical. Instead, the electron capture resulted primarily in the formation of a and y fragments, representing an alternative fragmentation pathway, likely initiated by the electron capture at a backbone amide nitrogen protonation site within the β amino acid residues.     

Two-step on-particle ionization/enrichment via a washing- and separation-free approach: multifunctional TiO2 nanoparticles as desalting, accelerating, and affinity probes for microwave-assisted tryptic digestion of phosphoproteins in ESI-MS and MALDI-MS: comparison with microscale TiO2

We introduce a simplified sample preparation method using bare TiO₂ nanoparticles (NPs) to serve as multifunctional nanoprobes (desalting, accelerating, and affinity probes) for effective enrichment of phosphopeptides from microwave-assisted tryptic digestion of phosphoproteins (α-casein, β-casein and milk) in Electrospray Ionization Mass Spectrometry (ESI-MS) and Matrix Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS). The results demonstrate that TiO₂ NPs can effectively enrich and accelerate the digestion reactions of phosphoproteins in aqueous solutions and also from complex real samples. After the microwave experiments, we directly injected the resulting solutions into the ESI-MS and MALDI-MS systems for analysis, and excellent sensitivity was achieved without the need for any washing procedure or separation process. The reasons are attributed to the high binding affinity and selectivity of TiO₂ NPs toward phosphopeptides. Thus, phosphopeptides can be adsorbed onto the TiO₂ NP surface. The digested or partially digested phosphoproteins can be concentrated onto the TiO₂ NP surface. This results in the effective or complete digestion of phosphoproteins in a short period of time (45 s). In addition, high sensitivity and sequence coverage of phosphopeptide can be obtained using TiO₂ NPs as microwave absorbers and affinity probes in MALDI-MS and ESI-MS. This is due to the photocatalytic nature of the TiO₂ NPs because the absorption of microwave radiation that can accelerate the activation of trypsin for efficient digestion of phosphoproteins and enhances the ionization of phosphopeptides. The lowest concentrations detected for ESI-MS and MALDI-MS were 0.1 μM and 10 fmol, respectively, for α-casein. Comparing the two-step approach of TiO₂ NPs with microscale TiO₂ particles, the microscale TiO₂ particles shows no effect on the microwave-assisted tryptic digestion of phosphoproteins. The current approach offers multiple advantages, such as great simplicity, high sensitivity and selectivity, straightforward and separation/washing-free technique for phosphorpeptide enrichment analysis.     

Absolute assignment of constitutional isomers via structurally diagnostic fragment ions: The challenging case of α- and β-acyl naphthalenes

A general mass spectrometric method is described for the absolute assignment of α- or β-acyl naphthalenes, via which the gaseous α- and β-naphthoyl cations of m/z 155 are used as structurally diagnostic fragment ions (SDFI). These stable acylium ions are common and normally abundant fragment ions of acylnaphthalenes in general. Using a pentaquadrupole mass spectrometer, CID experiments with argon and ion/molecule reactions with 2-methyl-1,3-dioxolane, isoprene, acetonitrile and propionitrile were performed but failed to distinguish the two SDFI. Reactions with ethyl vinyl ether and several homologues as well as ethyl vinyl thioether were, however, successful. In reactions with ethyl vinyl ether, the α-SDFI form a pair of diagnostic product ions of m/z 165 and m/z 181, which are absent in the corresponding spectrum of the β-SDFI. Methyl 4-(1-naphthyl)-2,4-dioxobutanoate was used as a test molecule for this class of constitutional isomers and absolute structural assignment as an α-acyl naphthalene was correctly performed via the characterization of its β-SDFI.     

Zirconium arsenate-modified silica nanoparticles for specific capture of phosphopeptides and direct analysis by matrix-assisted laser desorption/ionization mass spectrometry

In this paper, we report, as far as we are aware, the first use of zirconium arsenate-modified silica nanoparticles (ZrAs-SNPs) for specific capture of phosphopeptides, followed by matrix-assisted laser desorption/ionization mass spectrometric (MALDI MS) analysis. Under the optimized enrichment conditions, the efficiency and specificity of ZrAs-SNPs were evaluated with tryptic digests of four standard proteins (α-casein, β-casein, ovalbumin, and bovine serum albumin) and compared with those of titanium arsenate-modified silica nanoparticles (TiAs-SNPs). The results showed that more selective enrichment of multiply phosphorylated peptides was observed with ZrAs-SNPs than with TiAs-SNPs whereas TiAs-SNPs resulted in slightly better recovery of singly phosphorylated peptides. ZrAs-SNPs were chosen for direct capture of phosphopeptides from diluted human serum of healthy and adenocarcinoma individuals. Our experimental profiling of serum phosphopeptides revealed that the level of phosphorylated fibrinogen peptide A was up-regulated in the serum of adenocarcinoma patients in comparison with healthy adults. This suggests the possibility of using ZrAs-SNPs for discovery of biomarkers of the pathogenesis process of tumors.     

Use of polyethylenimine-modified magnetic nanoparticles for highly specific enrichment of phosphopeptides for mass spectrometric analysis

Phosphopeptides have been isolated and concentrated by use of polyethyleneimine (PEI)-modified magnetic nanoparticles as an extremely specific affinity probe. The particles specifically captured phosphopeptides from a tryptic digest of a protein mixture that contained 0.07% (mole/mole) phosphoproteins, which is the highest specificity obtained to date. The time required for enrichment of the phosphopeptides was 1 min only. PEI-modified magnetic nanoparticles carry positive charges over a wide range of pH—between 3 and 11. This feature means the particles are effectively dispersed in solution during phosphopeptide capture. Mass spectrometric analysis revealed the very high efficiency of enrichment of phosphopeptides that contain both single and multiply-phosphorylated sites. The detection limit in the analysis of phosphopeptides obtained from both bovine α-casein and β-casein by matrix-assisted laser desorption/ionization mass spectrometry was 5 fmol. This approach was also used to enrich the phosphopeptides in a protein digest obtained from non-fat milk.     

Apparent proton affinities of highly charged peptide ions

The apparent proton affinities (PA) of various charge states of three highly basic peptides [(KAP)₁₀, (KAP)₈, (KAA)₈] were measured by the “bracketing” method. For all three peptides the apparent PA decreases as the charge state increases and the magnitude of the decrease is consistent with an increase in coulombic repulsion in the highly protonated species. Based on a simple electrostatic model, theoretical PAs were predicted for each charge state and the values for (KAP)₁₀ and (KAP)₈ were within 10 kcal/mol of the experimental values. The maximum charge state of these peptides was observed in all cases even when the most volatile solvent was sufficiently basic to deprotonate that charge state in the gas phase. In solution (KAP)₈ exhibits a random coil secondary structure while (KAA)₈ exhibits an α-helix structure. Comparison of measured and calculated apparent PAs suggests that (KAP)₈ retains its solution random coil structure in the gas phase and (KAA)₈ retains the solution compact α-helix structure in the lower charge states but opens up to a β structure in the gas phase to minimize electrostatic repulsions in higher charge states.     

Monitoring the large-scale production of the antihypertensive peptides RYLGY and AYFYPEL by HPLC-MS

This work reports the quantitative analysis of two novel antihypertensive peptides α_s1-CN f(90-94), with sequence RYLGY, and α_s1-CN f(143-149), with sequence AYFYPEL, by high-performance liquid chromatography–mass spectrometry in food-grade hydrolysates of milk proteins. The method was validated and showed sufficient specificity, reproducibility, linearity and recovery. Linear calibrations of the molecular ions m/z 671.2 and 902.3 were selected for the determination of the peptides RYLGY and AYFYPEL, respectively, and showed good statistical results (R ² ≥ 0.995 and with no significant lack-of-fit). The simplicity of RP-HPLC-MS method allowed the automated quantification of both antihypertensive peptides without any sample pretreatment. The application of this method permitted the evaluation of some hydrolysis variables, i.e., substrate, temperature, hydrolysis time or enzyme/substrate ratio, on the formation of antihypertensive peptides. The quantitative analysis of RYLGY and AYFYPEL showed that ultrafiltration was not effective to improve the content in active peptides, containing the hydrolysates and their respective permeates similar peptide amounts.     

The highly selective capture of phosphopeptides by zirconium phosphonate-modified magnetic nanoparticles for phosphoproteome analysis

The highly selective capture of phosphopeptides from proteolytic digests is a great challenge for the identification of phosphoproteins by mass spectrometry. In this work, the zirconium phosphonate-modified magnetic Fe₃O₄/SiO₂ core/shell nanoparticles have been synthesized and successfully applied for the selective capture of phosphopeptides from complex tryptic digests of proteins before the analysis of MALDI-TOF mass spectrometry with the desired convenience of sample handling. The ratio of magnetic nanoparticle to protein and the incubation time for capturing phosphopeptides from complex proteolytic digests were investigated, and the optimized nanoparticle-to-protein ratio and incubation time were between 15:1 to 30:1 and 30 min, respectively. The excellent detection limit of 0.5 fmol β-casein has been achieved by MALDI-TOF mass spectrometry with the specific capture of zirconium phosphonate-modified magnetic Fe₃O₄ nanoparticles. The great specificity of zirconium phosphonate-modified magnetic Fe₃O₄ nanoparticles to phosphopeptides was demonstrated by the selective capture of phosphopeptides from a complex tryptic digest of the mixture of α-casein and bovine serum albumin at molar ratio of 1 to 100 in MALDI-TOF-MS analysis. An application of the magnetic nanoparticles to selective capture phosphopeptides from a tryptic digest of mouse liver lysate was further carried out by combining with nano-LC-MS/MS and MS/MS/MS analyses, and a total of 194 unique phosphopeptides were successfully identified.     

Antigenicity of heat-treated and trypsin-digested milk samples studied by an optical immunochip biosensor

Abstract  

Individuals with known hypersensitivity or food allergy need to avoid ingestion of provoking food. Correct labelling of allergenic content in manufactured food products and the reliable determination of its residual immunoreactivity after several processing steps are therefore a major concern for the food industry. We evaluated the applicability of a new immunochip biosensor system to reveal the allergenic profile of the whey protein β-lactoglobulin (β-LG) in its natural biological cow’s milk matrix upon processing by tryptic digestion and extensive heat treatment. Colorimetric immunochemical signals generated by gold nanoparticles (Au NPs), in particular their functional optical property based on resonance-enhanced absorption of mirror-reflected light, were directly visible to the ‘naked’ eye of the analyst without the need of any instrumentation or enzyme-substrate for read-out. By using affinity-purified polyclonal rabbit IgG against the native protein, no antigenicity was detected for tryptic fragments. Both heat-denatured whey proteins and cow’s whole milk, however, did not lose their antibody-binding capacity even after a processing time of 20 min at 95°C for the whey proteins, and 60 min at 90°C for the milk, though the immunochemical response was considerably low compared to the unprocessed β-LG. Additionally, cross-reactivity and the false positive as well as false negative predictive value of the chip system were highlighted critically.     

Gas-phase separations of complex tryptic peptide mixtures

High-resolution ion mobility and time-of-flight mass spectrometry techniques have been used to analyze complex mixtures of peptides generated from tryptic digestion of fourteen common proteins (albumin, bovine, dog, horse, pig, and sheep; aldolase, rabbit; β-casein, bovine; cytochrome c, horse; β–lactoglobulin, bovine; myoglobin, horse; hemoglobin, human, pig, rabbit, and sheep). In this approach, ions are separated based on differences in mobilities in helium in a drift tube and on differences in their mass-to-charge ratios in a mass spectrometer. From data recorded for fourteen individual proteins (over a m/z range of 405 to 1000), we observe 428 peaks, of which 205 are assigned to fragments that are expected from tryptic digestion. In a separate analysis, the fourteen mixtures have been combined and analyzed as one system. In the single dataset, we resolve 260 features and are able to assign 168 peaks to unique peptide sequences. Many other unresolved features are observed. Methods for assigning peptides based on the use of m/z information and existing mobilities or mobilities that are predicted by use of intrinsic size parameters are described. Received: 26 August 2000 / Revised: 15 November 2000 / Accepted: 16 November 2000     

Development of a New Clean-up Method for the Determination of 5-methyl-tetrahydrofolate in Milk Samples Using a Sol-Gel β-Lactoglobulin Column

The specific forms of folic acid, a vitamin of the B-group, are generally determined in foods after sample clean-up using affinity columns containing folate binding protein covalently bound to a solid support material. The new approach described in the present paper is to isolate the target analyte by using the less costly milk protein β-lactoglobulin, entrapped in the pores of a sol-gel glass. Sol-gel β-lactoglobulin columns were included in a clean-up procedure developed to determine 5-methyl-tetrahydrofolate in milk samples. Concentrations of 5-methyl-tetrahydrofolate in milk samples with different fat contents ranged from 31 to 70 ng/ml. Repeated use of the β-lactoglobulin columns for sample preparation resulted in a decrease of analyte recovery. However, due to the high column to column reproducibility good results were obtained when the standard addition method was performed with columns which had already been used for the same number of sample clean-ups.     

Molecular interpretation of the linear relationship between the entropy and the enthalpy of activation of charge transfer reactions in polar liquids

The well-known linear relationship (TΔS# =αΔH# +β, where 1 >α > 0,β > 0) between the entropy (ΔS#) and the enthalpy (ΔH#) of activation for reactions in polar liquids is investigated by using a molecular theory. An explicit derivation of this linear relation from first principles is presented for an outersphere charge transfer reaction. The derivation offers microscopic interpretation for the quantitiesα andβ. It has also been possible to make connection with and justify the arguments of Bell put forward many years ago     

The Hydrogen–Deuterium Exchange at α-Carbon Atom in <Emphasis Type="BoldItalic">N,N,N</Emphasis>-Trialkylglycine Residue: ESI-MS Studies

Derivatization of peptides as quaternary ammonium salts (QAS) is a known method for sensitive detection by electrospray ionization tandem mass spectrometry. Hydrogens at α-carbon atom in N,N,N-trialkylglycine residue can be easily exchanged by deuterons. The exchange reaction is base-catalyzed and is dramatically slow at lower pH. Introduced deuterons are stable in acidic aqueous solution and are not back-exchanged during LC-MS analysis. Increased ionization efficiency, provided by the fixed positive charge on QAS group, as well as the deuterium labeling, enables the analysis of trace amounts of peptides.