ANALYSIS OF PHOTO-OXIDIZED AMINO ACIDS IN TRYPTIC PEPTIDES OF CALF LENS γ-II CRYSTALLIN

Insoluble and crosslinked proteins and increased pigmentation in the eye lens are features of aging and cataracts. Determining the amino acids which are involved in insolubilization, crosslinking and visible light scattering will shed light on the mechanisms by which cataracts form. Calf lens γ-II crystallin was irradiated at 295 nm, digested and separated into tryptic peptides. Additional tryptic peptides were found in the digest of irradiated γ-II which were not present in the dark control digest. These peptides were identified by amino acid sequencing and shown to correspond to expected tryptic fragments of the protein, indicating more facile digestion in the UV-irradiated protein than in dark controls. Amino acid analysis of the irradiated protein and peptides showed losses of histidine. methionine and cysteine residues as compared to control samples. Tryptophan, which is not detected by amino acid analysis, was also found to be reactive since losses in its fluorescence intensity were observed after irradiation. Some of the photochemically active amino acids had lower than expected responses in amino acid sequencing experiments. This suggested specific sites of photochemical activity in the various peptides. The evidence for peptide crosslinks is also discussed.     

THE RATES OF PHOTOLYSIS OF THE FOUR INDIVIDUAL TRYPTOPHAN RESIDUES IN UV EXPOSED CALF γ-II CRYSTALLIN

Aqueous buffer solutions of the lens protein bovine gamma-II crystallin were irradiated at 295 nm in the presence of dithiothreitol to determine the individual photolysis susceptibilities of the four tryptophan residues. Reverse-phase high performance liquid chromatography was utilized to compare the tryptic peptide maps before and after irradiation. Sequence analysis of collected tryptic peptides showed that the four tryptophans in calf gamma-II crystallin. TRP-42, TRP-68, TRP-131, and TRP-157 appeared in four distinct tryptic peptides. Fluorescence and absorption (diode array) monitoring of the eluting peptides allowed assessment of the changes in peptide absorbance and fluorescence following irradiation. Tryptophan fluorescence losses of (40 +/- 15)%, (17 +/- 4)%, (35 +/- 5)% and (15 +/- 4)% were observed for the peptides containing TRP-42, TRP-68, TRP-131 and TRP-157, respectively. Thus the four tryptophans in calf gamma-II crystallin did not all photolyze at the same rate. The rate differences are presumably related to the microenvironments of the individual tryptophan residues, and this is discussed in terms of the known crystal structure of calf gamma-II crystallin.     

Matrix-assisted laser desorption/ionization mass spectrometric peptide mapping of high molecular weight glutenin subunits 1Bx7 and 1Dy10 in Cheyenne cultivar

This study describes the verification of the cDNA-deduced amino acid sequences of high molecular weight glutenin subunits 1Dy10 and 1Bx7 in Cheyenne cultivar by direct matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) analysis of their tryptic fragments omitting chromatographic pre-separation. These polypeptides have a conserved structure consisting of a long central repetitive domain that prevents the application of conventional sequencing procedures such as Edman degradation. The published sequence of subunit 1Dy10 contains 7 Lys and 13 Arg residues; thus the production of 21 tryptic peptides is expected. The cDNA-deduced sequence for 1Bx7 subunit includes 5 Lys and 15 Arg residues, but the presence of three Arg-Pro bonds, which are normally not cleaved by trypsin, predicts only 19 tryptic peptides. Three different matrices (DHB, SA and HCCA) in combination with the most compatible sample preparation procedures were used in order to obtain the maximum 1Dy10 and 1Bx7 sequence coverage. MALDI analysis of the 1Dy10 tryptic digest resulted in the identification of all 21 expected peptides. In the case of 1Bx7 MALDI analysis resulted in the identification of 17 of the 19 expected peptides, giving a sequence coverage of 99.3%. These results were sufficient to rule out glycosylation of the 1Dy10 and 1Bx7 proteins and to assess the absence of any other post-translational modification, to within the detection limits of the method.     

Tandem mass spectrometry of amidated peptides

The behavior of C-terminal amidated and carboxylated peptides upon low-energy collision-induced dissociation (CID) was investigated. Two sets of 76 sequences of variable amino acid compositions and lengths were synthesized as model compounds. In most cases, C-terminal amidated peptides were found to produce, upon CID, an abundant loss of ammonia from the protonated molecules. To validate such MS/MS signatures, the studied peptides contained amino acids that can potentially release ammonia from their side chains, such as asparagine, glutamine, tryptophan, lysine and arginine. Arginine, and to a lesser extent lysine, was shown to induce a competitive fragmentation leading to the loss of ammonia from their side chains, thus interfering with the targeted backbone neutral release. However, when arginine or lysine was located at the C-terminal position mimicking a tryptic digest, losses of ammonia from the arginine side chain and from the peptide backbone were completely suppressed. Such results were discussed in the frame of peptidomic or proteomic studies in an attempt to reveal the presence of C-terminal amidated peptides or proteins.     

Chemically-assisted fragmentation combined with multi-dimensional liquid chromatography and matrix-assisted laser desorption/ionization post source decay, matrix-assisted laser desorption/ionization tandem time-of flight or matrix-assisted laser desorption/ionization tandem mass spectrometry for improved sequencing of tryptic peptides

Derivatization of tryptic peptides using an Ettan CAF matrix-assisted laser desorption/ionization (MALDI) sequencing kit in combination with MALDI-post source decay (PSD) is a fast, accurate and convenient way to obtain de novo or confirmative peptide sequencing data. CAF (chemically assisted fragmentation) is based on solid-phase derivatization using a new class of water stable sulfonation agents, which strongly improves PSD analysis and simplifies the interpretation of acquired spectra. The derivatization is performed on solid supports, ZipTip(microC18, limiting the maximum peptide amount to 5 microg. By performing the derivatization in solution enabled the labeling of tryptic peptides derived from 100 microg of protein. To increase the number of peptides that could be sequenced, derivatized peptides were purified using multidimensional liquid chromatography (MDLC) prior to MALDI sequencing. Following the first dimension strong cation exchange (SCX) chromatography step, modified peptides were separated using reversed-phase chromatography (RPC). During the SCX clean up step, positively charged peptides are retained on the column while properly CAF-derivatized peptides (uncharged) are not. A moderately complex tryptic digest, prepared from six different proteins of equimolar amounts, was CAF-derivatized and purified by MDLC. Fractions from the second dimension nano RPC step were automatically sampled and on-line dispensed to MALDI sample plates and analyzed using MALDI mass spectrometry fragmentation techniques. All proteins in the derivatized protein mixture digest were readily identified using MALDI-PSD or MALDI tandem mass spectrometry (MS/MS). More than 40 peptides were unambiguously sequenced, representing a seven-fold increase in the number of sequenced peptides in comparison to when the CAF-derivatized protein mix digest was analyzed directly (no MDLC-separation) using MALDI-PSD. In conclusion, MDLC purification of CAF-derivatized peptides significantly increases the success rate for de novo and confirmative sequencing using various MALDI fragmentation techniques. This new approach is not only applicable to single protein digests but also to more complex digests and could, thus, be an alternative to electrospray ionization MS/MS for peptide sequencing.     

How to discriminate between leucine and isoleucine by low energy ESI-TRAP MS<Superscript>n</Superscript>

In peptide sequencing experiments involving a single step tandem mass acquisition, leucine and isoleucine are indistinguishable because both are characterized by a 113 Da mass difference from the other peptide fragments in the MS2 spectrum. In this work, we propose a new method to distinguish between these two amino acids in consecutive MSn experiments, exploiting a gas-phase fragmentation of isoleucine that leads to a diagnostic 69 Da ion. We used this method to assess the Leu/Ile residues of several synthetic peptides. The procedure was then tested on a tryptic digest of myoglobin, assigning the correct amino acid in the majority of the peptides. This work was performed with an old and low-resolution instrument, thus demonstrating that our method is suitable for a wide number of ion trap mass spectrometers, not necessarily expensive or up-to-date.     

Mass mapping analysis as a tool for the identification of genetic variants of bovine beta-casein

Matrix-assisted laser desorption ionisation time-of-flight mass spectrometric analysis of the tryptic digest of beta-casein A2 and beta-casein B was performed before and after the separation of the peptides by LC. The overlapping of the chromatograms showed that all peaks were present in both samples, except for one only found in the tryptic digest of the A2 variant and two in the B variant. Experimental masses could be assigned to those peptides produced by tryptic digest of beta-casein variant. This peptide mapping strategy and current methodological improvements represent a promising tool for the identification of milk genetic variants with the difference of an amino acid substitution.     

On-line amino acid-based capillary isoelectric focusing-ESI-MS/MS for protein digests analysis

Six amino acids with pIs that ranged from 3.2 to 9.7 were used as ampholytes to establish a pH gradient in capillary isoelectric focusing. This amino acid-based capillary isoelectric focusing (cIEF) was coupled with ESI-MS/MS using an electrokinetically pumped sheath-flow interface for peptide analysis. Amino acid-based isoelectric focusing generates a two-order of magnitude lower background signal than commercial ampholytes in the important m/z range of 300–1800. Good focusing was achieved for insulin receptor, which produced ∼10 s peak width. For 0.1 mg mL⁻¹ bovine serum albumin (BSA) digests, 24 ± 1 peptides (sequence coverage 47 ± 4%) were identified in triplicate analysis. As expected, the BSA peptides were separated according to their pI. The concentration detection limit for the BSA digests is 7 nM and the mass detection limit is 7 fmole. A solution of six bovine protein tryptic digests spanning 5 orders of magnitude in concentration was analyzed by amino acid based cIEF-ESI-MS/MS. Five proteins with a concentration range spanning 4 orders of magnitude were identified in triplicate runs. Using amino acid based cIEF-ESI-MS/MS, 112 protein groups and 303 unique peptides were identified in triplicate runs of a RAW 264.7 cell homogenate protein digest. In comparison with ampholyte based cIEF-ESI-MS/MS, amino acid based cIEF-ESI-MS/MS produces higher resolution of five acidic peptides, much cleaner mass spectra, and higher protein spectral counts.     

Gln-Gly cleavage: a dominant dissociation site in the fragmentation of protonated peptides

An understanding of the gas-phase dissociation of protonated peptides within the mass spectrometer is essential for automated high-throughput protein identification. In this communication we describe a facile cleavage of the Gln-Gly peptide bond under low-collisional energy conditions. A variety of synthetic peptides have been analysed where key amino acids have been substituted within the sequence PQGPPQQGGR, which is a consensus repeat present in the tryptic peptides of acidic proline-rich protein 1 (PRP-1). The collision-induced dissociation spectra obtained from the PRP-1 tryptic peptides and the synthetic peptides indicate that facile Gln-Gly cleavage occurs when an X-Gln-Gly-Y sequence is present in a peptide, where X is any amino acid and Y any amino acid other than Gly.     

Determination of the unusual amino acid hypusine at the lower picomole level by derivatization with 4-dimethylaminoazobenzene-4'-sulphonyl chloride and reversed-phase high-performance or medium-pressure liquid chromatography.

Hypusine, an unusual amino acid formed by post-translational modification of lysine, is normally determined by specific metabolic labelling followed by measurement of released radioactivity after protein hydrolysis. This paper describes a sensitive non-radioactive method for the determination of hypusine, involving complete protein hydrolysis and precolumn derivatization of the released amino acids with 4-dimethylaminoazobenzene-4'-sulphonyl chloride, followed by reversed-phase high-performance or medium-pressure liquid chromatography of the dabsylated derivatives. The detection limit of hypusine was about 500 fmol. Additionally, the hypusine-containing protein from the archaebacterium Sulfolobus acidocaldarius was purified. By applying the dabsylation method to the analysis of tryptic peptides derived from this protein, it was possible to determine the correct positioning of the hypusine residue in the amino acid sequence, which was not possible by the amino acid sequencing procedure alone.     

天花粉蛋白一级结构的修正及不同产地天花粉蛋白的研究

胰蛋白酶酶解天花粉蛋白, 用高效液相色谱分离酶解肽段, 用顺序仪测定其有关肽段的顺序。用羧肽酶A, B, Y测定了天花粉蛋白C-端和天花粉蛋白溴化氰降解肽CB1的C-端顺序, 修正了我们1985年测定的天花粉蛋白一级结构, 证明天花粉蛋白由246(7)氨基酸残基所组成, 除C-端微观不均一外, 与Collins结果一致。同时比较了芜湖产天花粉蛋白一级结构与平湖产的天花粉蛋白一级结构, 没有发现两者的一级结构有差别。     

THE MODIFICATION OF RIBONUCLEASE-A BY NEARULTRAVIOLET IRRADIATION IN THE PRESENCE OF PSORALEN

Abstract— Ribonuclease A is inactivated when irradiated under oxygen by UV-A light in the presence of psoralen. The rate of inactivation is greatly reduced by sodium azide. ascorbate or nitrogen, whereas the substrate gives only very limited protection. A ribonuclease sample modified to 40% remaining activity presented a significant modification of amino acid residues known to be sensitive to oxidation and 1.4 mol of bound psoralen per mol of protein. The secondary structure of the enzyme, as assessed by circular dicroism was not changed by irradiation; neither was aggregation of the enzyme to a higher mol wt evident. Studies on the tryptic peptides fractionated by high performance liquid chromatography showed that the photomodification occurs with very low selectivity. All the five peptides containing hystidine, tyrosine and methionine residues were greatly modified, although two, those containing histidine residues 12 and 119 in the sequence, amino acids known to be involved in the catalytic activity of ribonuclease. are modified to a greater extent. The protein bound psoralen. revealed by radioactivity in the HPLC eluate, was not found associated to only one or few peptide peaks but spread on a large zone of elution.     

Nanospray 'taxation' and how to avoid it

In mass spectrometric analysis with nanospray ionization, some analytes were found to appear in spectra with a delay of tens of minutes, while a few others could not be detected at all. The effect was found to be related to cation-exchange chromatography with negative charge on the glass surface, and with the most affected peptide or protein ions having strong localization of positive charge in blocks of two or more adjacent basic amino acid residues (e.g. melittin). The 'affinity' to the glass surface was studied with a peptide mixture and bovine serum albumin (BSA) tryptic digest solutions at sub-micromolar concentration. About 20% fewer tryptic peptides could be identified from spectra recorded with a glass nanospray capillary compared to those acquired with either conventional 1 microL/min electrospray or a quartz nanospray capillary. Protein identification studies are not likely to be seriously affected by this loss, but other protein applications, such as investigations of mutations or post-translational modifications, may suffer due to reduced sequence coverage. Ways to avoid losses of useful ions are discussed.     

Dynamic pH barrage junction focusing of amino acids,peptides, and digested monoclonal antibodies in capillary electrophoresis–mass spectrometry

Dynamic pH barrage junction focusing in CE enables effective signal enhancement, quantitative capture efficiencies, and straightforward optimization. The method is a technical variant of dynamic pH junction focusing. CE separation with dynamic pH barrage junction focusing is compatible with both optical and mass spectrometric detection. We developed a CE–MS/MS method using hydrophilic polyethyleneimine-coated capillaries and validated it for the qualitative analysis of amino acids, peptides, and tryptic peptides of digested monoclonal antibodies. The S/N of extracted ion electropherograms of zwitterionic analytes were enhanced by approximately two orders of magnitude with a tradeoff of a shortened separation window. Online focusing improved the MS signal intensity of a diluted antibody digest, enabling more precursor ions to be analyzed with subsequent tandem mass spectrometric identification. It also broadened the concentration range of protein digest samples for which adequate sequence coverage data can be obtained. With only 0.9 ng of digested infliximab sample loaded into the capillary, 76% and 100% sequence coverage was realized for antibody heavy and light chains, respectively, after online focusing. Full coverage was achieved with 9 ng of injected digest.     

Comparison of reversed-phase and weak anion-exchange high-performance liquid chromatographic methods for peptide separations

Weak anion-exchange and reversed-phase high-performance liquid chromatographic methods for peptide separations were compared using a tryptic digest of "rat small myelin basic protein". In these experiments, a number of tryptic peptides that were not resolved on the reversed-phase column could be separated on the weak anion-exchange column, and in other instances, as might be expected, reversed-phase chromatography provided better resolution of certain peptides than did the weak anion-exchange method. The results obtained strongly suggest that the combined use of these two methods of separation, which utilize different selectivities, can provide an excellent improvement in resolving power for a number of peptide separations.     

Identification of an artifact in the mass spectrometry of proteins derivatized with iodoacetamide

Derivatization of cysteinyl residues is often used to prevent the formation of disulfide bonds during protein isolation and analysis. The most commonly used reagents are iodoacetic acid and iodoacetamide, which increase the molecular mass of the protein by 58 or 57 Da, respectively, for each derivatized cysteine. A possible side reaction is derivatization of methionine. In our analysis of derivatized human lens alphaA-crystallins, we found an apparent molecular mass 48 Da lower than the mass expected for alphaA-crystallin with the cysteines carboxyamidomethylated. Analysis of a tryptic digest of this protein showed that both cysteines and one methionine had been derivatized. Peaks indicating a molecular mass 48 Da less than expected for the protein with only cysteines derivatized were attributed to fragmentation of the derivatized methionine through collision-induced dissociation in the electrospray ionization source. An awareness of this artifact is important to investigators searching for proteins and their modified forms in complex mixtures.     

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.     

Photooxidation of lens alpha-crystallin by hypericin (active ingredient in St. John's Wort)

Hypericin is the active ingredient in the over the counter antidepressant medication St. John's Wort. Hypericin produces singlet oxygen and other excited state intermediates that indicate it should be a very efficient phototoxic agent in the eye. Furthermore it absorbs in the UV and visible range, which means it can potentially damage both the lens and the retina. Lens alpha-crystallin, isolated from calf lenses, was irradiated in the presence of hypericin (5 x 10(-5) M, 10 mM ammonium bicarbonate, pH 7.0) and in the presence and absence of light (> 300 nm, 24 mW/cm2). Hypericin-induced photosensitized photopolymerization as assessed by sodium dodecylsulfate-polyacrylamide gel electrophoresis. Further analysis of the oxidative changes occurring in alpha-crystallin using mass spectrometry showed specific oxidation of methionine, tryptophan and histidine residues, which increased with irradiation time. Hypericin did not damage the lens protein in the dark. Damage to alpha-crystallin could undermine the integrity of the lens directly by protein denaturation and indirectly by disturbing chaperone function. Therefore, in the presence of light, hypericin can induce changes in lens protein that could lead to the formation of cataracts. Appropriate precautions should be taken to protect the eye from intense sunlight while on this antidepressant medication.     

Differential labeling of free and disulfide-bound thiol functions in proteins

A method for the simultaneous determination of the number of free cysteine groups and disulfide-bound cysteine groups in proteins has been developed based on the sequential labeling of free and bound thiol functionalities with two ferrocene-based maleimide reagents. Liquid chromatography/electrochemistry/mass spectrometry was used to assign the N-(2-ferroceneethyl)maleimide (FEM) labeled free cysteine functionalities in a tryptic digest mixture, whereas a precursor ion scan enables the detection of peptides with ferrocenecarboxylic acid-(2-maleimidoyl)ethylamide (FMEA) labeled disulfide-bound cysteine groups after reduction. Fragment spectra of the labeled peptides yield an excellent coverage of b-type and y-type ions. The ferrocene labeled cysteines were fragmented as 412 Da (FEM) and 455 Da (FMEA). These fragment masses are significantly higher than unlabeled amino acids or dipeptides and are easily detected. The position of free and disulfide-bound cysteine may therefore be assigned in an amino acid sequence.     

NEAR UV LIGHT AND CATARACTS*

Abstract. Sunlight and many types of artificial lighting contain near-UV light (300–400 nm). These wavelengths can enter the eye and are maximally absorbed in the lens due to its chemical composition. The lenses of certain animals develop cataracts from exposure to this light, and changes similar to those that occur in human lenses with certain types of cataracts and with aging are inducible in isolated human lenses. These changes seem to be associated with chemical alterations in the essential amino acid tryptophan either as a part of proteins or in free form. Such changes in tryptophan would result in lens cell toxicity, in increased pigmentation of the lens, and in large aggregates of proteins. The latter two changes would result in losses in the ability of the lens to transmit visible light needed for vision, and the abnormal state called cataract would result. Much more work is needed to prove that near-UV light can accelerate cataractous changes in the lenses of living humans. Studies at the basic chemical level are needed, but population studies would be most essential for the final proof. Many preventive measures could become available, including the use of special types of spectacles and dietary additives.