Simultaneous determination and validated quantification of human insulin and its synthetic analogues in human blood serum by immunoaffinity purification and liquid chromatography-mass spectrometry

Possible fatal complications of human insulin and its synthetic analogues like hypoglycemia require precise classification and quantitative determination of these drugs both for clinical purposes as well as for forensic toxicologists. A procedure was developed for the identification and quantification of human insulin and different long-acting as well as short-acting synthetic insulins in human blood serum specimens. After an immunoaffinity purification step and separation by liquid chromatography, the insulins were characterized by their five- or sixfold protonated molecule ions and diagnostic product ions. Clinical samples of 207 diabetic and 50 non-diabetic patients after the administration of human insulin or oral antidiabetics and forensic samples were analyzed for human/synthetic insulin concentrations. The method was validated according to international guidelines. Limits of detection of the insulins ranged between 1.3 and 2.8?μU/ml. Recoveries ranged between 33.2?% and 51.7?%. Precision data was in accordance with international guidelines. Clinical samples showed concentrations of human insulin lower than 301?μU/ml. Our liquid chromatography tandem mass spectrometry procedure allows unambiguous identification and quantification of the intact human insulin and its intact synthetic analogues Humalog?, Novolog?, Apidra?, Lantus?, and Levemir? in human blood serum in clinical and overdose cases. The assay could be successfully tested in patients with diabetes mellitus on therapy with insulins or oral antidiabetics.     

Charge state separation for protein applications using a quadrupole time-of-flight mass spectrometer

A novel method for separating ions according to their charge state using a quadrupole time-of-flight mass spectrometer is presented. The benefits of charge state separation are particularly apparent in protein identification applications at low femtomole concentration levels, where in conventional TOF MS spectra peptide ions are often lost in a sea of chemical noise. When doubly and triply charged tryptic peptide ions need to be filtered from singly charged background ions, the latter are suppressed by two to three orders of magnitude, while from 10-50% of multiply charged ions remain. The suppression of chemical noise reduces the need for chromatography and can make this experimental approach the electrospray equivalent of conventional MALDI peptide maps. If unambiguous identification cannot be achieved, MS/MS experiments are performed on the precursor ions identified through charge separation, while the previously described Q2-trapping duty cycle enhancement is tuned for approximately 1.4 of the precursor m/z to enhance intensities of ions with m/z values above that of the precursor. The resulting product ion spectra contain few fragments of impurities and provide quick and unambiguous identification through database search. The multiple charge separation technique requires minimal tuning and may become a useful tool for analysis of complex mixtures.     

Matrix-assisted laser desorption/ionization directed nano-electrospray ionization tandem mass spectrometric analysis for protein identification

In those cases where the information obtained by peptide mass fingerprinting or matrix-assisted laser desorption/ionization tandem mass spectrometry (MALDI-MS/MS) is not sufficient for unambiguous protein identification, nano-electrospray ionization (nano-ESI) and/or electrospray ionization tandem mass spectrometry (ESI-MS/MS) analysis must be performed. The sensitivity of nano-ESI/MS, however, is lower than that of MALDI-MS, especially at very low analyte concentrations and/or in the presence of contaminants, such as salt and detergents. Moreover, to perform ESI-MS/MS, the peptide masses of the precursor ions must be known. The approach described in this paper, MALDI-directed nano-ESI-MS/MS, makes use of information obtained from the more sensitive MALDI-MS experiments in order to direct subsequent nano-ESI-MS/MS experiments. Peptide molecular ions found in the MALDI-MS analysis are then selected, as their (+2) precursor ions, for nano-ESI-MS/MS sequencing, even though these ions cannot be detected in the ESI-MS spectra. This method, originally proposed by Tempst et al. (Anal. Chem. 2000, 72: 777-790), has been extended to provide better sensitivity and shorter analysis times; also, a comparison with liquid chromatography/tandem mass spectrometry (LC/MS/MS) has been performed. These experiments, performed using quadrupole time-of-flight instruments equipped with commercially available nano-ESI sources, have allowed the unambiguous identification of in-gel digested proteins at levels below their ESI-MS detection limits, even in the presence of salts and detergents.     

Separation and identification of organic acid-coenzyme A thioesters using liquid chromatography/electrospray ionization-mass spectrometry

A method has been developed for the direct determination of coenzyme A (CoA) and organic acid-CoA thioesters in mixtures using directly combined liquid chromatography/electrospray ionization-mass spectrometry (LC/ESI-MS). Mixtures of CoA and organic acid-CoA thioesters were analyzed by LC/ESI-MS with detection of protonated molecular ions and characteristic fragment ions for each compound. The identities of the CoA-thioesters were established based on LC retention times and simultaneously recorded mass spectra. Monitoring of the CoA specific fragment ion at m/z 428 throughout the chromatogram provides a unique fingerprint for CoA content in the samples that corroborates the identification of organic acid-CoA thioesters in the mixtures. Furthermore, fragment ions arising from the ester linkage portion of the molecule allow unambiguous identification of the CoA esters in the samples. A second LC elution system was developed that allows the simultaneous separation and identification of 2-hydroxypropionyl-CoA (lactyl-CoA) and 3-hydroxypropionyl CoA (3HP-CoA), which have the same mass and identical MS fragmentation behavior. The utility of LC/ESI-MS employing this elution system is demonstrated by the determination of 3HP-CoA and lactyl-CoA (converted to CoA-thioesters from their corresponding free acids using CoA-transferase) in fermentation broths from Escherichia coli strains engineered for the production of 3-hydroxypropionic acid (3HP). External calibration employing a purified 3HP-CoA standard allowed indirect quantification of 3HP content in the broth with a precision of 1% (RSD). The feasibility of extending the method described above to perform LC/selected reaction monitoring-tandem mass spectrometry for direct determination of organic acid-CoA thioesters in cells was also demonstrated.     

Identification of components in waste streams by electrospray and tandem mass spectrometry

Highly polar, non-gas-chromatographable compounds have few unambiguous analysis protocols for environmental applications. A recent environmental investigation, concerning the identification of a non-gas-chromatographable yellow component in chemical waste water and in effluents from a biological wastewater treatment plant required the use of a number of analytical approaches. Electrospray mass spectrometry, tandem mass spectrometry, high-performance liquid chromatography, nuclear magnetic resonance, and molecular spectroscopy of commercial and synthesized chlorodinitrophenol isomers were required in order to identify the specific isomer causing the color. The present report summarizes the electrospray ionization and tandem mass spectrometric studies that were used. The mass spectrometric study shows that two different isomers of chlorodinitrophenol exhibit very different collision-induced dissociation (CID) spectra. Differences in the tandem mass spectra can be attributed to the different structures of the anions formed from these two different isomers. Instrumentation that uses electrospray ionization and produces CID mass spectra and optical absorption spectra in a single analysis may be required in order to produce highly specific information on non-gas-chromatographable compounds found in the environment.     

Identification and characterization of the chemical constituents of Simiao Wan by ultra high performance liquid chromatography with mass spectrometry coupled to an automated multiple data processing method

The chemical constituents of Simiao Wan (SW), a traditional Chinese medicine preparation, are difficult to determine and remain unclear. To more efficiently detect ions, a multiple data processing approach has been used in the characterization of the compounds. In this study, a rapid and sensitive method based on ultra high performance liquid chromatography with mass spectrometry and the multiple data processing approach was established to characterize the chemical constituents of SW. Ultra high performance liquid chromatography with mass spectrometry coupled with the multiple data processing approach could efficiently remove nonrelated ion signals from accurate mass data. We report the application of the multiple data processing approach for comprehensive detection and rapid identification of chemical constituents of SW. Of note, the total analysis time for separation was less than 20 min without losing any resolution. In the variable, importance in projection plot of orthogonal projection to latent structure‐discriminant analysis, a total of 72 ions of interest (37 ions in positive mode, 38 ions in negative mode and three ions in both mode) were extracted or tentatively characterized based on their retention times, exact mass measurement for each molecular ion and subsequent fragment ions. In summary, the methodology proposed in this study could be valuable for the structural characterization and identification of the multiple constituents in the traditional Chinese medicine formula SW.     

Simultaneous quantitation of 17 female sex hormones in illegal products by validated liquid chromatography-high resolution mass spectrometry and liquid chromatography-tandem mass spectrometry methods

The consumption of food and drugs adulterated with female sex hormones can have an extremely adverse effect on human health. Therefore, developing appropriate monitoring methods for the identification of various exogenous female sex hormones is crucial for minimizing and eliminating the related health risks. Herein, 17 female hormones categorized into two groups: estrogen and progestin, were analyzed using reversed-phase liquid chromatography coupled to Orbitrap or triple quadrupole mass spectrometry. The fragmentation patterns for all compounds were discovered, and fragmented structures were also derived from them through liquid chromatography–high-resolution mass spectrometry followed by qualitative sample analysis. In addition, a quantitative analysis of 67 samples of illicit drugs and dietary supplements was performed using the validated liquid chromatography-tandem mass spectrometry method. Female hormone components were detected in two samples of an unauthorized injectable solution and a tablet-type drug. Medroxyprogesterone was detected in the samples in the range of 96.4–206 ng/g. Notably, eight components similar in structure to steroids were simultaneously detected as male sex hormones by confirming their fragmentation ion patterns using liquid chromatography–high-resolution mass spectrometry. The developed methods thus offer a dependable and practically applicable approach for the screening and detection of exogenous female sex hormones in real food and drug samples to ensure public health.     

The use of library identification and common fragments for the identification of corticosteroids in forensic samples

The detection of corticosteroids and sex steroids in samples with no content indication, which are confiscated for forensic investigation, is a challenge in doping analysis. A screening method based on the identification of androgens, estrogens, gestagens, and their esters by means of a mass spectral library, along with a fast ultra-performance liquid chromatography–mass spectrometry (UPLC-MS) method, was recently developed in our lab for the analysis of dietary supplements. However, for forensic investigations, it is important to extend the scope of the method to corticosteroids in various matrices. Therefore, 36 corticosteroids were added to the mass spectral library, and the sample preparation step was modified so that androgens, gestagens, corticosteroids, and their esters could be analyzed with only one injection with the UPLC-MS method. A complementary tool to the existing library identification was found in the extraction of common fragment ions out of the full scan data obtained for the library search. The fragment ion with m/z 147 was found to be a good marker for the detection of steroids. Extra confirmation was obtained from the fragment ions with m/z 135 (for all steroids) and 237 (specific for corticosteroids) or from the fragment ions with m/z 77, 91, and 105. The effectiveness of this approach was evaluated on some samples previously screened for forensic investigation with thin-layer chromatography and confirmed with a targeted gas chromatography–mass spectrometry method. This study shows that the combination of the library identification and the common fragment ions approach can be a valuable tool in the detection of steroids without defining any target at the start of the analysis.     

Analysis of sucralose and other sweeteners in water and beverage samples by liquid chromatography/time-of-flight mass spectrometry

A methodology for the chromatographic separation and analysis of three of the most popular artificial sweeteners (aspartame, saccharin, and sucralose) in water and beverage samples was developed using liquid chromatography/time-of-flight mass spectrometry (LC/TOF-MS). The sweeteners were extracted from water samples using solid-phase extraction (SPE) cartridges. Furthermore, several beverages were analyzed by a rapid and simple method without SPE, and the presence of the sweeteners was confirmed by accurate mass measurements below 2-ppm error. The unambiguous confirmation of the compounds was based on accurate mass measurements of the protonated molecules [M+H]⁺, their sodium adducts and their main fragment ions. Quantitation was carried out using matrix-matched standard calibration and linearity of response over 2 orders of magnitude was demonstrated (r > 0.99). A detailed fragmentation study for sucralose was carried out by time-of-flight and a characteristic spectrum fingerprint pattern was obtained for the presence of this compound in water samples. Finally, the analysis of several wastewater, surface water and groundwater samples from the US showed that sucralose can be found in the aquatic environment at concentrations up to 2.4 μg/L, thus providing a good indication of wastewater input from beverage sources.     

Trace mycotoxin analysis in complex biological and food matrices by liquid chromatography-atmospheric pressure ionisation mass spectrometry

Mycotoxins are toxic secondary metabolites produced by filamentous fungi that are growing on agricultural commodities. Their frequent presence in food and their severe toxic, carcinogenic and estrogenic properties have been recognised as potential threat to human health. A reliable risk assessment of mycotoxin contamination for humans and animals relies basically on their unambiguous identification and accurate quantification in food and feedstuff. While most screening methods for mycotoxins are based on immunoassays, unambiguous analyte confirmation can be easily achieved with mass spectrometric methods, like gas chromatography/mass spectrometry (GC/MS) or liquid chromatography/mass spectrometry (LC/MS). Due to the introduction of atmospheric pressure ionisation (API) techniques in the late 80s, LC/MS has become a routine technique also in food analysis, overcoming the traditional drawbacks of GC/MS regarding volatility and thermal stability. During the last few years, this technical and instrumental progress had also an increasing impact on the expanding field of mycotoxin analysis. The aim of the present review is to give an overview on the application of LC-(API)MS in the analysis of frequently occurring and highly toxic mycotoxins, such as trichothecenes, ochratoxins, zearalenone, fumonisins, aflatoxins, enniatins, moniliformin and several other mycotoxins. This includes also the investigation of some of their metabolites and degradation products. Suitable sample pre-treatment procedures, their applicability for high sample through-put and their influence on matrix effects will be discussed. The review covers literature published until July 2006.     

Determination of biogenic amines in food samples using derivatization followed by liquid chromatography/mass spectrometry

A liquid chromatography (LC)/mass spectrometry method was developed for the determination of selected biogenic amines in various fish and other food samples. It is based on a precolumn derivatization of the amines with succinimidylferrocenyl propionate under formation of the respective amides and their reversed-phase liquid-chromatographic separation with subsequent electrospray ionization mass-spectrometric detection. Deuterated putescine, cadaverine, and histamine are added prior to the derivatization as internal standards that are coeluted, thus allowing excellent reproducibility of the analysis to be achieved. Depending on the analyte, the limits of detection were between 1.2 and 19.0 mg/kg, covering between 2 and 3 decades of linearity. The limit of detection and the linear range for histamine are suitable for the surveillance of the only defined European threshold for biogenic amines in fish samples. Compared with the established ortho-phthalaldehyde (OPA)/LC/fluorescence method, the newly developed method allows an unambiguous identification of the biogenic amines by their mass spectra in addition to only retention times, a fivefold acceleration of the separation, and independency from the sample matrix owing to the isotope-labeled internal standards. Various fish, calamari, and salami samples were successfully analyzed with the new method and validated with an independent OPA/LC/fluorescence method.     

Fast analysis of doping agents in urine by ultra-high-pressure liquid chromatography–quadrupole time-of-flight mass spectrometry. II: Confirmatory analysis

For doping control, analyses of samples are generally achieved in two steps: a rapid screening and, in the case of a positive result, a confirmatory analysis. A two-step methodology based on ultra-high-pressure liquid chromatography coupled to a quadrupole time-of-flight mass spectrometry (UHPLC–QTOF-MS) was developed to screen and confirm 103 doping agents from various classes (e.g., β-blockers, stimulants, diuretics, and narcotics). The screening method was presented in a previous article as part I (i.e., Fast analysis of doping agents in urine by ultra-high-pressure liquid chromatography–quadrupole time-of-flight mass spectrometry. Part I: screening analysis). For the confirmatory method, basic, neutral and acidic compounds were extracted by a dedicated solid-phase extraction (SPE) in a 96-well plate format and detected by MS in the tandem mode to obtain precursor and characteristic product ions. The mass accuracy and the elemental composition of precursor and product ions were used for compound identification. After validation including matrix effect determination, the method was considered reliable to confirm suspect results without ambiguity according to the positivity criteria established by the World Anti-Doping Agency (WADA). Moreover, an isocratic method was developed to separate ephedrine from its isomer pseudoephedrine and cathine from phenylpropanolamine in a single run, what allowed their direct quantification in urine.     

Multi-residue method for the analysis of 101 pesticides and their degradates in food and water samples by liquid chromatography/time-of-flight mass spectrometry

A comprehensive multi-residue method for the chromatographic separation and accurate mass identification of 101 pesticides and their degradation products using liquid chromatography/time-of-flight mass spectrometry (LC/TOF-MS) is reported here. Several classes of compounds belonging to different chemical families (triazines, organophosphorous, carbamates, phenylureas, neonicotinoids, etc.) were carefully chosen to cover a wide range of applications in the environmental field. Excellent chromatographic separation was achieved by the use of narrow accurate mass windows (0.05 Da) in a 30 min interval. Accurate mass measurements were always below 2 ppm error for all the pesticides studied. A table compiling the accurate masses for 101 compounds together with the accurate mass of several fragment ions is included. At least the accurate mass for one main fragment ion for each pesticide was obtained to achieve the minimum of identification points according to the 2002/657/EC European Decision, thus fulfilling the EU point system requirement for identification of contaminants in samples. The method was validated with vegetable samples. Calibration curves were linear and covered two orders of magnitude (from 5 to 500 microg/L) for most of the compounds studied. Instrument detection limits (LODs) ranged from 0.04 to 150 microg/kg in green-pepper samples. The methodology was successfully applied to the analysis of vegetable and water samples containing pesticides and their degradation products. This paper serves as a guide for those working in the analytical field of pesticides, as well as a powerful tool for finding non-targets and unknowns in environmental samples that have not been previously included in any of the routine target multi-residue methods.     

Determination of nerve agent degradation products in environmental samples by liquid chromatography-time-of-flight mass spectrometry with electrospray ionization

A powerful and rapid method has been developed for the identification and quantitative determination of alkyl methylphosphonic acids, which are the degradation products of nerve agents, using liquid chromatography-time-of-flight mass spectrometry with electrospray ionization. Six alkyl methylphosphonic acids were well separated within 16 min. For quantitative analysis, good linearity, sensitivity and reproducibility were obtained by LC-MS in the selected ion monitoring mode. For unambiguous identification of alkyl methylphosphonic acids, fragment ions were produced by in-source collision induced dissociation (CID), and then exact mass measurement of CID fragment ions was performed. The feasibility of applying this technique for detecting these compounds in spiked environmental waters and soils was demonstrated.     

Isotope abundance analysis methods and software for improved sample identification with supersonic gas chromatography/mass spectrometry

We present newly developed isotope abundance analysis (IAA) methods and software which are used to derive elemental formula information from experimental mass spectral data of molecular ion isotopomeric abundances. The software, using a novel method, can also be used to automatically confirm or reject NIST library search results, thereby significantly improving the confidence level in sample identifications. In the case of IAA confirmation of the NIST library results, sample identification is unambiguous, since the confirmation is achieved by two independent sets of data and analytical methods. In the case of a rejection, such as when the molecule is not included in the library's databases, the IAA software independently provides a list of elemental formulae with declining order of matching to the isotopomeric experimental data, in a similar way to accurate mass measurements with costly instruments. IAA is ideally applicable to gas chromatography/mass spectrometry (GC/MS) (and liquid chromatography/electron ionization mass spectrometry (LC/EI-MS)) with a supersonic molecular beam (SMB) since it requires a trustworthy and highly abundant true molecular ion that is unique to the SMB-MS systems, plus the absence of self chemical ionization and vacuum background noise, again unique features of GC/SMB-MS. The various features of the IAA methods and software are described, their performance is demonstrated with the analysis of experimental GC-SMB-MS data and the IAA concept is compared with accurate mass alternatives. The combination of IAA and GC/SMB-MS is believed to be superior to accurate mass GC/MS in view of the general availability of trustworthy molecular ions for an extended range of compounds.     

Versatile lipid profiling by liquid chromatography–high resolution mass spectrometry using all ion fragmentation and polarity switching. Preliminary application for serum samples phenotyping related to canine mammary cancer

Lipids represent an extended class of substances characterized by such high variety and complexity that makes their unified analyses by liquid chromatography coupled to either high resolution or tandem mass spectrometry (LC–HRMS or LC–MS/MS) a real challenge. In the present study, a new versatile methodology associating ultra high performance liquid chromatography coupled to high resolution tandem mass spectrometry (UHPLC–HRMS/MS) have been developed for a comprehensive analysis of lipids. The use of polarity switching and “all ion fragmentation” (AIF) have been two action levels particularly exploited to finally permit the detection and identification of a multi-class and multi-analyte extended range of lipids in a single run. For identification purposes, both higher energy collision dissociation (HCD) and in-source CID (collision induced dissociation) fragmentation were evaluated in order to obtain information about the precursor and product ions in the same spectra. This approach provides both class-specific and lipid-specific fragments, enhancing lipid identification. Finally, the developed method was applied for differential phenotyping of serum samples collected from pet dogs developing spontaneous malignant mammary tumors and health controls. A biological signature associated with the presence of cancer was then successfully revealed from this lipidome analysis, which required to be further investigated and confirmed at larger scale.     

New and automated MS<Superscript>n</Superscript> approaches for top-down identification of modified proteins

An automated top-down approach including data-dependent MS(3) experiment for protein identification/characterization is described. A mixture of wild-type yeast proteins has been separated on-line using reverse-phase liquid chromatography and introduced into a hybrid linear ion trap (LTQ) Fourier transform ion cylclotron resonance (FTICR) mass spectrometer, where the most abundant molecular ions were automatically isolated and fragmented. The MS(2) spectra were interpreted by an automated algorithm and the resulting fragment mass values were uploaded to the ProSight PTM search engine to identify three yeast proteins, two of which were found to be modified. Subsequent MS(3) analyses pinpointed the location of these modifications. In addition, data-dependent MS(3) experiments were performed on standard proteins and wild-type yeast proteins using the stand alone linear trap mass spectrometer. Initially, the most abundant molecular ions underwent collisionally activated dissociation, followed by data-dependent dissociation of only those MS(2) fragment ions for which a charge state could be automatically determined. The resulting spectra were processed to identify amino acid sequence tags in a robust fashion. New hybrid search modes utilized the MS(3) sequence tag and the absolute mass values of the MS(2) fragment ions to collectively provide unambiguous identification of the standard and wild-type yeast proteins from custom databases harboring a large number of post-translational modifications populated in a combinatorial fashion.     

Detection and identification of alkylphosphonic acids by positive electrospray ionization tandem mass spectrometry using a tricationic reagent

The retrospective detection and identification of degradation products of chemical warfare agents are of immense importance in order to prove their spillage and use. A highly sensitive liquid chromatography/electrospray ionization tandem mass spectrometric (LC/ESI-MS/MS) method--using an imidazolium-based tricationic reagent--was developed for the detection and identification of the anionic degradation products of nerve agents. A commercially available solution of 1,3-imidazolium-bis-(1-hexylbenzylimidazolium) trifluoride (IBHBI) formed adducts with alkylphosphonic acids (APAs), allowing detection of the APAs by positive mode ESI-MS. Tandem mass spectrometry was used for the unambiguous identification of the APAs. Parameters influencing the formation and stability of these adduct during mass spectrometric analysis, such as solvent composition, concentration of IBHBI, effect of pH and interferences by salts, were optimized. The absolute limits of detection (0.1 ng) for achieved for the APAs were better than those previously reported, and linear dynamic ranges of 10-2000 ng mL(-1) were achieved. The method was repeatable with a relative standard deviation ≤7.3%. APAs present in aqueous samples provided by the Organization for the Prohibition of Chemical Weapons during the 22(nd) and 24(th) Official Proficiency tests were detected and identified as IBHBI adducts. The added advantage of this method is that low-mass analytes are detected at higher mass, thus obviating the problem with background noise at low mass.     

Quantitative 252Cf plasma desorption mass spectrometry for pharmaceuticals: A new approach to coupling liquid chromatography with mass spectrometry

A ²⁵²Cf plasma desorption mass spectrometer has been set up to analyse thin layers of non-volatile organic samples. Molecular and fragment ions were produced and their mass was determined by a time-of-flight measurement. A novel interface combines a high-performance liquid chromatograph with the ²⁵²Cf plasma desorption mass spectrometer in a twofold way: introducing the effluent continuously through a capillary inlet in the on-line liquid chromatography mass spectrometry mode or transferring already prepared samples through a vacuum lock into the mass spectrometer in the off-line liquid chromatography+mass spectrometry mode. The off-line mode has been applied for the quantitative analysis of pharmaceuticals in blood using stable isotope labelled standards.     

Determination of Abnormal Hemoglobins by the Combined use of Reversed-Phase high Performance Liquid Chromatography and Fast Atom Bombardment Mass Spectrometry

Abstract

Separation by reversed-phase high performance liquid chromatography (RP-HPLC) of peptide mixtures obtained from tryptic digestion of abnormal human hemoglobins and subsequent analysis by fast atom bombardment mass spectrometry (FAB-MS) of selected peptides allow unambiguous, rapid identification of the hemoglobin variants. By this method two varied hemoglobins, Hb D Punjab and Hb J Sardegna, have been determined. The methodology described, which is simple and reliable, could be used for the detection and structural identification of known and unknown Hb variants, without amino acid (a.a.) or sequence analysis.