低浓度甲醛对多肽和蛋白化学修饰的质谱研究

采用基质辅助激光解析电离飞行时间质谱( MALDI-TOF MS)和纳升电喷雾四极杆飞行时间串联质谱( Nano-ESI -QTOF MS)技术,以标准肽段和流感病毒基质蛋白酶切肽段为模型,研究了甲醛对蛋白质和多肽主链的修饰作用。采用与实际病毒灭活过程一致的实验条件(4℃,0.025%(V/V)福尔马林(37%(w/w)甲醛溶液)处理72 h),进行甲醛与多肽的化学反应。结果表明,在实验条件下,甲醛能与标准肽段N端的氨基反应生成羟甲基加合物,再发生缩合反应生成亚胺,形成+12 Da的产物。此外,甲醛还能与标准肽段中的精氨酸、赖氨酸的侧链发生反应,生成+12 Da的反应产物。对流感病毒基质蛋白的酶切肽段与甲醛的反应的质谱分析结果显示,多数的肽段都生成了+24 Da的产物,质量的增加来源于肽段N端氨基(+12 Da)和C端精氨酸或赖氨酸的侧链(+12 Da)的贡献。此外,还观察到有一个漏切位点的肽段生成了+36 Da的产物。本研究结果表明,在实验条件下,低浓度甲醛主要与肽段和蛋白的N 端氨基,以及精氨酸和赖氨酸侧链发生反应。本研究为分析低浓度甲醛与蛋白质的反应产物提供了有效的质谱分析方法和解谱依据。     

Mass spectrometric characterization of lipid-modified peptides for the analysis of acylated proteins

The analysis of acylated proteins by mass spectrometry (MS) has largely been overshadowed in proteomics by the analysis of glycosylated and phosphorylated proteins; however, lipid modifications on proteins are proving to be of increasing importance in biomedical research. In order to identify the marker ions and/or neutral loss fragments that are produced upon collision-induced dissociation, providing a means to identify the common lipid modifications on proteins, peptides containing an N-terminally myristoylated glycine, a palmitoylated cysteine and a farnesylated cysteine were chemically synthesized. Matrix-assisted laser desorption/ionization time-of-flight time-of-flight (MALDI-TOF-TOF), electrospray ionization quadrupole time-of-flight (ESI Q-TOF), and electrospray ionization hybrid triple-quadrupole/linear ion trap (ESI QqQ(LIT)) mass spectrometers were used for the analysis. The peptide containing the N-terminally myristoylated glycine, upon CID, produced the characteristic fragments a1 (240.4 Th) and b1 (268.4 Th) ions as well as a low-intensity neutral loss of 210 Da (C14H26O). The peptides containing a farnesylated cysteine residue fragmented to produce a marker ion at a m/z of 205 Th (C15H25) as well as other intense farnesyl fragment ions, and a neutral loss of 204 Da (C15H24). The peptides containing a palmitoylated cysteine moiety generated neutral losses of 238 Da (C16H30O) and 272 Da (C16H32OS); however, no marker ions were produced. The neutral losses were more prominent in the MALDI-TOF-TOF spectra, whereas the marker ions were more abundant in the ESI QqQ(LIT) and Q-TOF mass spectra.     

Efficient identification and quantification of proteins using isotope-coded 1-(6-methylnicotinoyloxy)succinimides by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

We describe a convenient and useful method for the identification and relative quantification of proteins using light and heavy reagents, 1-(6-methylnicotinoyloxy)succinimides (6-CH(3)-Nic-NHS and 6-CD(3)-Nic-NHS, respectively). This method is based on the chemical derivatization of amino groups of tryptic peptides with these reagents, i.e., the basic moiety of the reagents thus incorporated into both the N-terminal amino group and the epsilon-amino group of the lysine residue would improve the ionization efficiency of tryptic peptides. An increase in protein sequence coverage is achieved by derivatization with these reagents or by combination of mass values before and after derivatization. Since a combination of 6-CH(3)-Nic-NHS and d(3)-labeled reagent (6-CD(3)-Nic-NHS) generates a 3 Da mass difference per reaction site, the d(3)-labeled reagent shifts the mass values of d(0)-labeled peptides according to the number of reactive amino groups in the peptides. In the case of tryptic peptides, the mass values of C-terminal arginine and lysine peptides are shifted by 3 and 6 Da, respectively. Further, the 3 Da mass difference between 6-CH(3)-Nic-NHS and 6-CD(3)-Nic-NHS offers a means for the relative quantification of protein by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.     

Fragmentation of cationized phosphotyrosine containing peptides by atmospheric pressure MALDI/Ion trap mass spectrometry

An investigation of phosphate loss from sodium-cationized phosphotyrosine containing peptide ions was conducted using liquid infrared (2.94 microm) atmospheric pressure matrix-assisted laser desorption/ionization (AP MALDI) coupled to an ion trap mass spectrometer (ITMS). Previous experiments in our laboratory explored the fragmentation patterns of protonated phosphotyrosine containing peptides, which experience a loss of 98 Da under CID conditions in the ITMS. This loss of 98 Da is unexpected for phosphotyrosine, given the structure of its side chain. Phosphate loss from phosphotyrosine residues seems to be dependent on the presence of arginine or lysine residues in the peptide sequence. In the absence of a basic residue, the protonated phosphotyrosine peptides do not undergo losses of HPO(3) (Delta 80 Da) nor HPO(3) + H(2)O (Delta 98 Da) in their CID spectra. However, sodium cationized phosphotyrosine containing peptides that do not contain arginine or lysine residues within their sequences do undergo losses of HPO(3) (Delta 80 Da) and HPO(3) + H(2)O (Delta 98 Da) in their CID spectra.     

Photodissociation of singly protonated peptides at 193 nm investigated with tandem time-of-flight mass spectrometry

Photodissociation at 193 nm of some singly protonated peptides generated by matrix-assisted laser desorption/ionization was investigated using tandem time-of-flight mass spectrometry. For peptides with arginine at the C-terminus, x, upsilon, and w fragment ions were generated preferentially while a and d fragment ions dominated for peptides with arginine at the N-terminus. These are the same characteristics as photodissociation at 157 nm reported previously. Overall, the photodissociation spectra obtained at 157 and 193 nm were strikingly similar.     

Electrospray ionization quadrupole time-of-flight and matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometric analyses to solve micro-heterogeneity in post-translationally modified peptides from Phoneutria nigriventer (Aranea, Ctenidae) venom

Previous studies of the fractionated venom of the Brazilian armed spider Phoneutria nigriventer, obtained by gel filtration, have demonstrated the presence of a fraction PhM, a pool of small peptides (up to 2000 Da) that provoke contractions in smooth muscle of guinea pig ileum. Initial attempts to sequence these peptides were largely unsuccessful because of the low purification yield and the fact that the majority seemed to be blocked at their N-termini. In the present work, analysis of this venom fraction by mass spectrometry has revealed the existence of a highly complex mixture of peptides with molecular weights corresponding to those observed for the muscle-active peptides previously described (800-1800 Da). These peptides appear to be a family of isoforms with some particular features. The amino acid sequences of 15 isoforms have been determined by tandem mass spectrometry (MS/MS) using both electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-Q/ToFMS) and matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry (MALDI-ToF/ToFMS). These molecules contain post-translational modifications such as proteolysis and C-terminal amidation, which combine to generate additional isoforms. All the isoforms sequenced in this study possess an N-terminal pyroglutamic acid residue. A search for sequence similarities with other peptides in databanks revealed that these peptides are structurally related to the tachykinins, a family of neuro-hormone peptides. The data obtained in this study will be essential for the subsequent steps of this research, the synthesis of these peptides and pharmacological characterization of their biological activity.     

Fragmentation of phosphopeptides by atmospheric pressure MALDI and ESI/ion trap mass spectrometry

An investigation of phosphate loss from phosphopeptide ions was conducted, using both atmospheric pressure matrix-assisted laser desorption/ionization (AP MALDI) and electrospray ionization (ESI) coupled to an ion trap mass spectrometer (ITMS). These experiments were carried out on a number of phosphorylated peptides in order to investigate gas phase dephosphorylation patterns associated with phosphoserine, phosphothreonine, and phosphotyrosine residues. In particular, we explored the fragmentation patterns of phosphotyrosine containing peptides, which experience a loss of 98 Da under collision induced dissociation (CID) conditions in the ITMS. The loss of 98 Da is unexpected for phosphotyrosine, given the structure of its side chain. The fragmentation of phosphoserine and phosphothreonine containing peptides was also investigated. While phosphoserine and phosphothreonine residues undergo a loss of 98 Da under CID conditions regardless of peptide amino acid composition, phosphate loss from phosphotyrosine residues seems to be dependent on the presence of arginine or lysine residues in the peptide sequence.     

Collision-induced dissociation of lys-lys intramolecular crosslinked peptides

The use of chemical crosslinking is an attractive tool that presents many advantages in the application of mass spectrometry to structural biology. The correct assignment of crosslinked peptides, however, is still a challenge because of the lack of detailed fragmentation studies on resultant species. In this work, the fragmentation patterns of intramolecular crosslinked peptides with disuccinimidyl suberate (DSS) has been devised by using a set of versatile, model peptides that resemble species found in crosslinking experiments with proteins. These peptides contain an acetylated N-terminus followed by a random sequence of residues containing two lysine residues separated by an arginine. After the crosslinking reaction, controlled trypsin digestion yields both intra- and intermolecular crosslinked peptides. In the present study we analyzed the fragmentation of matrix-assisted laser desorption/ionization-generated peptides crosslinked with DSS in which both lysines are found in the same peptide. Fragmentation starts in the linear moiety of the peptide, yielding regular b and y ions. Once it reaches the cyclic portion of the molecule, fragmentation was observed to occur either at the following peptide bond or at the peptide crosslinker amide bond. If the peptide crosslinker bond is cleaved, it fragments as a regular modified peptide, in which the DSS backbone remains attached to the first lysine. This fragmentation pattern resembles the fragmentation of modified peptides and may be identified by common automated search engines using DSS as a modification. If, on the other hand, fragmentation happens at the peptide bond itself, rearrangement of the last crosslinked lysine is observed and a product ion containing the crosslinker backbone and lysine (m/z 222) is formed. The detailed identification of fragment ions can help the development of softwares devoted to the MS/MS data analysis of crosslinked peptides.     

A tandem time-of-flight mass spectrometer: combination of a multi-turn time-of-flight and a quadratic-field mirror

A tandem time-of-flight (ToF) mass spectrometer consisting of a multi-turn time-of-flight (ToF) and a quadratic-field ion mirror has been designed and constructed. The instrument combines the unique capabilities of both ToF instruments, namely high-resolution and monoisotopic precursor ion selection from the multi-turn ToF and temporal focus for fragment ions with different kinetic energies from the quadratic-field mirror. The first tandem mass spectra for this unique combination of ToF systems are presented.     

Mass spectrometric identification of in vivo carbamylation of the amino terminus of Ectothiorhodospira mobilis high-potential iron-sulfur protein,isozyme 1

The complete amino acid sequence of a novel high-potential iron-sulfur protein (HiPIP) isozyme 1 from the moderately halophilic phototrophic bacterium Ectothiorhodospira mobilis was determined by a combined approach of chemical and mass spectrometric sequencing techniques. By mass analysis of the apo- and holo-protein in the positive electrospray ionization mode using different electrospray solvents, the protein was found to be post-translationally modified by a moiety of 43 Da. Further analysis showed the nature and location of this modification to be a carbamyl group at the N-terminus of the HiPIP. This rare type of modification has previously been reported to occur in the water-soluble human lens alphaB-crystallin, class D beta-lactamases and some prokaryotic ureases, albeit at an internal lysine residue. In this paper, we discuss the mass spectrometric features of a carbamylated residue at the N-terminus of a peptide or a lysine side-chain during sequence analysis by collision-induced dissociation tandem mass spectrometry. Our data provide evidence for the first case of a prokaryotic carbamylated electron transport protein occurring in vivo.     

Analysis of high mass peptides using a novel matrix-assisted laser desorption/ionisation quadrupole ion trap time-of-flight mass spectrometer

A novel matrix-assisted laser desorption/ionisation quadrupole ion trap time-of-flight (MALDI QIT ToF) mass spectrometer has been used to analyse high mass peptide ions exceeding 2000 Da. Human adrenocorticotropic hormone (fragment 18-39) and oxidised bovine insulin chain B were utilised to evaluate the performance of the instrument both in MS and in MS/MS mode. Its ability to efficiently isolate ions and to fragment them using collisionally activated decomposition (CAD) has been demonstrated using mixtures diluted to the low-femtomole level on target. Additionally, multiple stage mass spectrometry (MS/MS/MS) provides a second-generation product ion spectrum in which new fragment ions are detected and new stretches of amino acids are identified.     

Arginine-mimic labeling with guanidinoethanethiol to increase mass sensitivity of lysine-terminated phosphopeptides by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) generally shows better mass sensitivity for arginine-terminated peptides than for lysine-terminated peptides, presumed to arise from the higher proton affinity of the guanidine group in arginine. Here, we report a new method for analyzing phosphopeptides in which phosphopeptides are labeled with a novel chemical tag, guanidinoethanethiol (GET), by a beta-elimination/Michael addition before MS analysis. GET labeling converts phosphoserine into guanidinoethylcysteine (Gec) containing a guanidine moiety, along with an increase in mass of 21.1 Da. GET-labeled peptides are detected by MALDI MS with greatly increased peak intensities compared to those of intact phosphopeptides. In particular, GET labeling of lysine-terminated phosphopeptides dramatically increased peak intensity. GET labeling of lysine-terminated phosphopeptides improved sensitivity up to 22 times compared to that of the corresponding aminoethanethiol (AET) labeling, in which AET was used as a labeling tag containing an amino group instead of the guanidine group. These results show the guanidine group plays a very important role in increasing the observed sensitivity of MALDI MS for labeled peptide, derivatized from serine-phosphorylated peptides.     

Neutral loss of amino acid residues from protonated peptides in collision-induced dissociation generates N- or C-terminal sequence ladders

The widespread occurrence of the neutral loss of one to six amino acid residues as neutral fragments from doubly protonated tryptic peptides is documented for 23 peptides with individual sequences. Neutral loss of amino acids from the N-terminus of doubly charged tryptic peptides results in doubly charged y-ions, forming a ladder-like series with the ions [M + 2H](2+) = y(max) (2+), y(max - 1) (2+), y(max - 2) (2+), etc. An internal residue such as histidine, proline, lysine or arginine appears to favor this type of fragmentation, although it was sometimes also observed for peptides without this structure. For doubly protonated non-tryptic peptides with one of these residues at or near the N-terminus, we observed neutral loss from the C-terminus, resulting in a doubly charged b-type ion ladder. The analyses were performed by Q-TOF tandem mass spectrometry, facilitating the recognition of neutral loss ladders by their 2+ charge state and the conversion of the observed mass differences into reliable sequence information. It is shown that the neutral loss of amino acid residues requires low collision offset values, a simple mechanistic explanation based on established fragmentation rules is proposed and the utility of this neutral loss fragmentation pathway as an additional source for dependable peptide sequence information is documented.     

Proteome profiling of human cerebrospinal fluid: exploring the potential of capillary electrophoresis with surface modified capillaries for analysis of complex biological samples

A bottom-up proteomic approach, based on capillary electrophoresis (CE) in combination with matrix- assisted laser desorption/ionization tandem time-of-flight mass spectrometry (MALDI-ToF/ToF MS), was used to analyze immunoaffinity depleted human cerebrospinal fluid (CSF) and compare it with a non-depleted sample. After enzymatic digestion and desalting, the tryptic peptides were separated by CE using PolyE-323 modified capillaries and fractionated off-line onto MALDI target plates for further analysis by MALDI-MS and MS/MS. The protein profile of the depleted sample was compared with non depleted CSF. Overall, 85 proteins were identified with 95% significance in both samples. The significance scores for proposed biomarkers, such as amyloid-like protein 1 precursor, could be increased up to 12 times after the depletion. Other proteins, often suggested to be related to neurodegenerative diseases, like amyloid beta A4 protein precursor, superoxide dismutase and apolipoprotein E precursor could only be found in the depleted CSF samples. The effect of a derivatization of tryptic peptides with 2- methoxy-4,5-dihydro-1H-imidazole reagent for protein identification with MS was also employed to increase the number of identified proteins and the sequence coverages. The results presented in this study illustrate the benefit of combining a sample pre-fractionation step and a label's ability to enhance the ionization efficiency with the potential of CE using PolyE-323 modified capillaries in the analysis of complex samples. The straight-forward approach that provides speed and simplicity resulting in high-resolution separations and low sample consumption represents an easily applicable separation technique that can serve as a complement to other currently existing analytical approaches needed in modern proteomic analysis of clinically relevant samples.     

Determination of pyridoxal‐5′‐phosphate (PLP)‐bonding sites in proteins: a peptide mass fingerprinting approach based on diagnostic tandem mass spectral features of PLP‐modified peptides

Peptides modified by pyridoxal‐5′‐phosphate (PLP), linked to a lysine residue via reductive amination, exhibit distinct spectral characteristics in the collision‐induced dissociation (CID) tandem mass (MS/MS) spectra that are described here. The MS/MS spectra typically display two dominant peaks whose m/z values correspond to neutral losses of [H₃PO₄] (?98 Da) and the PLP moiety as [C₈H₁₀NO₅P] (?231 Da) from the precursor peptide ion, respectively. Few other peaks are observed. Recognition of this distinct fragmentation behavior is imperative since determining sequences and sites of modifications relies on the formation of amide backbone cleavage products for subsequent interpretation via proteome database searching. Additionally, PLP‐modified peptides exhibit suppressed precursor ionization efficiency which diminishes their detection in complex mixtures. Presented here is a protocol which describes an enrichment strategy for PLP‐modified peptides combined with neutral loss screening and peptide mass fingerprinting to map the PLP‐bonding site in a known PLP‐dependent protein. This approach represents an efficient alternative to site‐directed mutagenesis which has been the traditional method used for PLP‐bonding site localization in proteins. Copyright © 2009 John Wiley & Sons, Ltd.     

Atmospheric pressure matrix-assisted laser desorption/ionisation ion trap mass spectrometry of synthetic polymers: a comparison with vacuum matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry

Atmospheric pressure matrix-assisted laser desorption/ionisation quadrupole ion trap (AP-MALDI/QIT) mass spectrometry has been investigated for the analysis of polyethylene glycol (PEG 1500) and a hyperbranched polymer (polyglycidol) in the presence of alkali-metal salts. Mass spectra of PEG 1500 obtained at atmospheric pressure showed dimetallated matrix/analyte adducts, in addition to the expected alkali-metal/PEG ions, for all matrix/alkali-metal salt combinations. The relative intensities of the desorbed ions were dependent on the matrix, the alkali-metal salt added to aid cationisation and the ion trap interface conditions [capillary temperature, in-source collisionally-induced dissociation (CID)]. These data indicate that the adducts are rapidly stabilised by collisional cooling enabling them to be transferred into the ion trap. Experiments using identical sample preparation conditions were carried out on a vacuum MALDI time-of-flight (ToF) mass spectrometer. In all cases, vacuum MALDI-ToF spectra showed only alkali-metal/PEG ions and no matrix/analyte adducts. The tandem mass spectrometry (MS/MS) capability of the ion trap has been demonstrated for a lithiated polyglycol yielding a rich fragment-ion spectrum. Analysis of the hyperbranched polymer polyglycidol by AP-MALDI/QIT reveals the characteristic ion series for these polymers as also observed under vacuum MALDI-ToF conditions.     

Study of an Unusual Advanced Glycation End-Product (AGE) Derived from Glyoxal Using Mass Spectrometry

Glycation is a post-translational modification (PTM) that affects the physiological properties of peptides and proteins. In particular, during hyperglycaemia, glycation by α-dicarbonyl compounds generate α-dicarbonyl-derived glycation products also called α-dicarbonyl-derived advanced glycation end products. Glycation by the α-dicarbonyl compound known as glyoxal was studied in model peptides by MS/MS using a Fourier transform ion cyclotron resonance mass spectrometer. An unusual type of glyoxal-derived AGE with a mass addition of 21.98436 Da is reported in peptides containing combinations of two arginine-two lysine, and one arginine-three lysine amino acid residues. Electron capture dissociation and collisionally activated dissociation results supported that the unusual glyoxal-derived AGE is formed at the guanidino group of arginine, and a possible structure is proposed to illustrate the 21.9843 Da mass addition.

Characterization and de novo sequencing of Atlantic salmon vitellogenin protein by electrospray tandem and matrix-assisted laser desorption/ionization mass spectrometry

Vitellogenin (VTG) is a protein produced by the liver of oviparous animals in response to circulating estrogens. In the plasma of males and immature females, VTG is undetectable. VTG has been used as a biomarker for exposure to endocrine disruptors in many species. In the present study, characterization of intact Atlantic salmon VTG was effected using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI ToF MS). Tryptic digest peptides were analyzed by MALDI ToF MS to obtain a peptide mass fingerprint. De novo sequencing of the tryptic peptides used low-energy collisionally-induced dissociation (CID) in an electrospray ionization quadrupole-ToF orthogonal hybrid mass spectrometer (ESI Q-ToF MS/MS). The interpretation of the product-ion spectra obtained from the ESI Q-ToF MS/MS was done by Lutefisk, a computer-based software algorithm. The molecular mass of the intact protein was found to be 187335 Da. A total of 14 tryptic peptides were sequenced and compared with the complete rainbow trout VTG and the partial Atlantic salmon VTG sequences found in the Swiss-Prot database. De novo sequencing by CID MS/MS of 11 Atlantic salmon tryptic digest peptides with selected precursor ions at m/z 788.24, 700.20, 794.75, 834.31, 889.28, 819.79, 865.27, 843.81, 572.20, 573.66 and 561.68 showed high homology with the known sequence of rainbow trout VTG. The last two precursor peptide ions, found at m/z 573.66 and m/z 561.68, also specifically matched the known portion of the Atlantic salmon VTG sequence. Finally, three tryptic precursor peptide ions found at m/z 795.18, 893.28 and 791.05, provided product-ion spectra, which were exclusive to the unsequenced portion of the Atlantic salmon VTG.     

A picomole-scale method for rapid peptide sequencing through convenient and efficient N-terminal phosphorylation and electrospray ionization mass spectrometry

Free or resin-bound peptides were phosphorylated at their N-termini by reacting with dimethyl phosphite in the presence of tetrachloromethane and triethylamine, and some of them were labeled using partial deuterium-labeled dimethyl phosphites (molar ratio of m + 6 and m = 1:1 or m + 6, m + 3 and m = 1:2:1) as the phosphorylating agents. The monophosphorylation of the lysine-containing peptides selectively occurred on the amino group of the N-terminus, not the side-chain of lysine residue. The resin-bound phosphoramidate peptides were cleaved by TFA before ESI-MS. The modified peptides were determined by electrospray ionization mass spectrometry, the protonated molecules of the unlabeled phosphoramidate peptides showed the singlet peaks, and those of the labeled phosphoramidate peptides displayed the doublet and/or triplet peaks. Tandem mass spectra (MS/MS) of the chosen protonated molecules gave sequential loss of the amino acid residues from the C-termini of the peptides, providing a convenient and rapid method for peptide sequencing.     

Rapid determination of amino acid incorporation by stable isotope labeling with amino acids in cell culture (SILAC)

Stable isotope labeling with amino acids in cell culture (SILAC) has evolved to be a major technique for quantitative proteomics using cell cultures. We developed a rapid method to follow and determine the incorporation of arginine and lysine. Analysis of the heavy state is required to avoid quantification errors. Moreover, the mixture of light and heavy states can be exploited to normalize the protein amount for subsequent relative quantification experiments. Therefore, peptides from different cell lines were extracted with 0.1% trifluoroacetic acid and analyzed by matrix-assisted laser desorption/ionization tandem time-of-flight (MALDI-TOF/TOF) mass spectrometry (MS). This analysis was highly reproducible and was performed in less than 2 h, significantly faster than other methods for the same purpose. Similar peptide mass profiles were obtained for human EBV-transformed B, Jurkat T, and HeLa cells as well as for mouse embryonic fibroblasts. Proteolytic fragments of 27 human proteins were identified with 56 peptides by MALDI-MS/MS and can be used as a database for these kinds of experiments. Sequencing revealed that the peptides were predominantly amino- and carboxy-terminal protein fragments displaying a specificity characteristic of the acidic proteases cathepsin D and E. Many of the identified peptides contained arginine and/or lysine, allowing determination of the incorporation rate of these amino acids. Furthermore, the rate of conversion of arginine into proline could be monitored easily.