Real Time Normalization of Fast Photochemical Oxidation of Proteins Experiments by Inline Adenine Radical Dosimetry.

Hydroxyl radical protein footprinting (HRPF) is a powerful method for measuring protein topography, allowing researchers to monitor events that alter the solvent accessible surface of a protein (e.g., ligand binding, aggregation, conformational changes, etc.) by measuring changes in the apparent rate of reaction of portions of the protein to hydroxyl radicals diffusing in solution. Fast Photochemical Oxidation of Proteins (FPOP) offers an ultrafast benchtop method for radical generation for HRPF, photolyzing hydrogen peroxide using a UV laser to generate high concentrations of hydroxyl radicals that are consumed on roughly a microsecond time scale. The broad reactivity of hydroxyl radicals means that almost anything added to the solution (e.g., ligands, buffers, excipients, etc.) will scavenge hydroxyl radicals, altering their half-life and changing the effective radical concentration experienced by the protein. Similarly, minute changes in peroxide concentration, laser fluence, and buffer composition can alter the effective radical concentration, making reproduction of data challenging. Here, we present a simple method for radical dosimetry that can be carried out as part of the FPOP workflow, allowing for measurement of effective radical concentration in real time. Additionally, by modulating the amount of radical generated, we demonstrate that effective hydroxyl radical yields in FPOP HRPF experiments carried out in buffers with widely differing levels of hydroxyl radical scavenging capacity can be compensated on the fly, yielding statistically indistinguishable results for the same conformer. This method represents a major step in transforming FPOP into a robust and reproducible technology capable of probing protein structure in a wide variety of contexts.

Towards high-throughput fast photochemical oxidation of proteins: Quantifying exposure in high fluence microtiter plate photolysis.

Protein structural analysis by mass spectrometry has gained significant popularity in recent years, including high-resolution protein topographical mapping by fast photochemical oxidation of proteins (FPOP). The ability to provide protein topographical information at moderate spatial resolution makes FPOP an attractive technology for the protein pharmaceutical discovery and development processes. However, current technology limits the throughput and requires significant manual sample manipulation. Similarly, as FPOP is being used on larger samples, sample flow through the capillary becomes challenging. No systematic comparison of the performance of static flash photolysis with traditional flow FPOP has been reported. Here, we evaluate a 96-well microtiter-based laser flash photolysis method for the topographical probing of proteins, which subsequently could be used to analyze higher order structure of the protein in a high-throughput fashion with minimal manual sample manipulation. We used multiple metrics to compare microtiter FPOP performance with that of traditional flow FPOP: adenine-based hydroxyl radical dosimetry, oxidation efficiency of a model peptide, and hydroxyl radical protein footprint of myoglobin. In all cases, microtiter plate FPOP performed comparably with traditional flow FPOP, requiring a small fraction of the time for exposure. This greatly reduced sample exposure time, coupled with automated sample handling in 96-well microtiter plates, makes microtiter-based FPOP an important step in achieving the throughput required to adapt hydroxyl radical protein footprinting for screening purposes.

Reduced plasminogen binding and delayed activation render γ'-fibrin more resistant to lysis than γA-fibrin.

Fibrin (Fn) clots formed from γ'-fibrinogen (γ'-Fg), a variant with an elongated γ-chain, are resistant to lysis when compared with clots formed from the predominant γA-Fg, a finding previously attributed to differences in clot structure due to delayed thrombin-mediated fibrinopeptide (FP) B release or impaired cross-linking by factor XIIIa. We investigated whether slower lysis of γ'-Fn reflects delayed plasminogen (Pg) binding and/or activation by tissue plasminogen activator (tPA), reduced plasmin-mediated proteolysis of γ'-Fn, and/or altered cross-linking. Clots formed from γ'-Fg lysed more slowly than those formed from γA-Fg when lysis was initiated with tPA/Pg when FPA and FPB were both released, but not when lysis was initiated with plasmin, or when only FPA was released. Pg bound to γ'-Fn with an association rate constant 22% lower than that to γA-Fn, and the lag time for initiation of Pg activation by tPA was longer with γ'-Fn than with γA-Fn. Once initiated, however, Pg activation kinetics were similar. Factor XIIIa had similar effects on clots formed from both Fg isoforms. Therefore, slower lysis of γ'-Fn clots reflects delayed FPB release, which results in delayed binding and activation of Pg. When clots were formed from Fg mixtures containing more than 20% γ'-Fg, the upper limit of the normal level, the delay in lysis was magnified. These data suggest that circulating levels of γ'-Fg modulate the susceptibility of clots to lysis by slowing Pg activation by tPA and provide another example of the intimate connections between coagulation and fibrinolysis.

The action of MBL-associated serine protease 1 (MASP1) on factor XIII and fibrinogen.

The complement system is an important recognition and effector mechanism of the innate immune system that upon activation leads to the elimination of foreign bodies. It can be activated through three pathways of which the lectin pathway is one. The lectin pathway relies on the binding of mannan-binding lectin (MBL) or the ficolins and the subsequent activation of the MBL-associated serine proteases (MASPs), namely, MASP1, 2 and 3 which all form complexes with both MBL and the ficolins. Major substrates have only been identified for MASP2 i.e. C4 and C2. For MASP1 only a few protein substrates which are cleaved at a low rate have been identified while none are known for MASP3. Since chromogenic substrate screenings have shown that MASP1 has thrombin-like activity, we wanted to investigate the catalytic potential of MASP1 towards two major proteins involved in the clotting process, fibrinogen and factor XIII, and compare the activity directly with that of thrombin. We found that rMASP1 and thrombin cleave factor XIII A-chain and the fibrinogen beta-chain at identical sites, but differ in cleavage of the fibrinogen alpha-chain. The thrombin turnover rate of factor XIII is approximately 650 times faster than that of rMASP1 at 37 degrees C, pH 7.4. rMASP1 cleavage of fibrinogen leads to the release of the proinflammatory peptide fibrinopeptide B. Thus rMASP1 has similar, but not identical specificity to thrombin and its catalytic activity for factor XIII and fibrinogen cleavage is much lower than that of thrombin. Nevertheless, rMASP1 can drive the formation of cross-linked fibrinogen. Since MASP1 is activated on contact of MBL or the ficolins with microorganisms, fibrinogen and factor XIII may be involved in the elimination of invading pathogens.

Protein ladder sequencing.

A new approach to protein sequencing is described. It consists of two steps: (i) ladder-generating chemistry, the controlled generation from a polypeptide chain by wet chemistry of a family of sequence-defining peptide fragments, each differing from the next by one amino acid; and (ii) data readout, a one-step readout of the resulting protein sequencing ladder by matrix-assisted laser-desorption mass spectrometry. Each amino acid was identified from the mass difference between successive peaks, and the position in the data set defined the sequence of the original peptide chain. This method was used to directly locate a phosphoserine residue in a phosphopeptide. The protein ladder sequencing method lends itself to very high sample throughput at very low per cycle cost.

Nanostructured diamond-like carbon on digital versatile disc as a matrix-free target for laser desorption/ionization mass spectrometry.

A nanostructured diamond-like carbon (DLC) coated digital versatile disk (DVD) target is presented as a matrix-free sample support for application in laser desorption/ionization mass spectrometry (LDI-MS). A large number of vacancies, defects, relative sp(2) carbon content, and nanogrooves of DLC films support the LDI phenomenon. The observed absorptivity of DLC is in the range of 305-330 nm (nitrogen laser, 337 nm). The universal applicability is demonstrated through different analytes like amino acids, carbohydrates, lipids, peptides, and other metabolites. Carbohydrates and amino acids are analyzed as sodium and potassium adducts. Peptides are detectable in their protonated forms, which avoid the extra need of additives for ionization. A bovine serum albumin (BSA) digest is analyzed to demonstrate the performance for peptide mixtures, coupled with the material-enhanced laser desorption/ionization (MELDI) approach. The detection limit of the described matrix-free target is investigated to be 10 fmol/microL for [Glu(1)]-fibrinopeptide B (m/z 1570.6) and 1 fmol/microL for L-sorbose (Na(+) adduct). The device does not require any chemical functionalization in contrast to other matrix-free systems. The inertness of DLC provides longer lifetimes without any deterioration in the detection sensitivity. Broad applicability allows high performance analysis in metabolomics and peptidomics. Furthermore the DLC coated DVD (1.4 GB) sample support is used as a storage device for measured and processed data together with sampling on a single device.

Role of 'B-b' knob-hole interactions in fibrin binding to adsorbed fibrinogen.

BACKGROUND: The formation of a fibrin clot is supported by multiple interactions, including those between polymerization knobs 'A' and 'B' exposed by thrombin cleavage and polymerization holes 'a' and 'b' present in fibrinogen and fibrin. Although structural studies have defined the 'A-a' and 'B-b' interactions in part, it has not been possible to measure the affinities of individual knob-hole interactions in the absence of the other interactions occurring in fibrin. OBJECTIVES: We designed experiments to determine the affinities of knob-hole interactions, either 'A-a' alone or 'A-a' and 'B-b' together. METHODS: We used surface plasmon resonance to measure binding between adsorbed fibrinogen and soluble fibrin fragments containing 'A' knobs, desA-NDSK, or both 'A' and 'B' knobs, desAB-NDSK. RESULTS: The desA- and desAB-NDSK fragments bound to fibrinogen with statistically similar K(d)'s of 5.8 +/- 1.1 microm and 3.7 +/- 0.7 microm (P = 0.14), respectively. This binding was specific, as we saw no significant binding of NDSK, which has no exposed knobs. Moreover, the synthetic 'A' knob peptide GPRP and synthetic 'B' knob peptides GHRP and AHRPY, inhibited the binding of desA- and/or desAB-NDSK. CONCLUSIONS: The peptide inhibition findings show both 'A-a' and 'B-b' interactions participate in desAB-NDSK binding to fibrinogen, indicating 'B-b' interactions can occur simultaneously with 'A-a'. Furthermore, 'A-a' interactions are much stronger than 'B-b' because the affinity of desA-NDSK was not markedly different from desAB-NDSK.

B:b interactions are essential for polymerization of variant fibrinogens with impaired holes 'a'.

BACKGROUND: Fibrin polymerization is mediated by interactions between knobs 'A' and 'B' exposed by thrombin cleavage, and holes 'a' and 'b' always present in fibrinogen. The role of A:a interactions is well established, but the roles of knob:hole interactions A:b, B:b or B:a remain ambiguous. OBJECTIVES: To determine whether A:b or B:b interactions have a role in thrombin-catalyzed polymerization, we examined a series of fibrinogen variants with substitutions altering holes 'a': gamma364Ala, gamma364His or gamma364Val. METHODS: We examined thrombin- and reptilase-catalyzed fibrinopeptide release by high-performance liquid chromatography, fibrin clot formation by turbidity, fibrin clot structure by scanning electron microscopy (SEM) and factor (F) XIIIa-catalyzed crosslinking by sodium dodecylsulfate polyacrylamide gel electrophoresis. RESULTS: Thrombin-catalyzed fibrinopeptide A release was normal, but fibrinopeptide B release was delayed for all variants. The variant fibrinogens all showed markedly impaired thrombin-catalyzed polymerization; polymerization of gamma364Val and gamma364His were more delayed than gamma364Ala. There was absolutely no polymerization of any variant with reptilase, which exposed only knobs 'A'. SEM showed that the variant clots formed after 24 h had uniform, ordered fibers that were thicker than normal. Polymerization of the variant fibrinogens was inhibited dose-dependently by the addition of either Gly-Pro-Arg-Pro (GPRP) or Gly-His-Arg-Pro (GHRP), peptides that specifically block holes 'a' and 'b', respectively. FXIIIa-catalyzed crosslinking between gamma-chains was markedly delayed for all the variants. CONCLUSION: These results demonstrate that B:b interactions are critical for polymerization of variant fibrinogens with impaired holes 'a'. Based on these data, we propose a model wherein B:b interactions participate in protofibril formation.

Identification of a new truncated form and deamidation products of fibrinopeptide B released by thrombin from human fibrinogen.

Quantification of fibrinopeptides release is widely used to investigate fibrinogen activation, and standard chromatographic or capillary electrophoretic procedures are readily available. However, in the analyses of fibrinopeptide mixtures derived from the action of thrombin on human fibrinogen, a few unidentified peaks are usually present. The composition of these peaks was studied by reverse-phase HPLC/MS, revealing a single major anomalous peptide having a molecular mass of 1384.4. A further MS/MS analysis allowed the identification of this form, as a Nterminally truncated fibrinopeptide B (fpB) lacking the first two residues (pyroglutamic acid and glycine). This previously unidentified, relatively low-abundance form ( approximately 7%) has been found consistently in our fibrinopeptides preparations, and analysis of the parent Bbeta-chain suggest that it is likely present in circulating fibrinogen. In addition, deamidated forms of all fpB species (including desArgB), resulting from the conversion of asparagine to aspartic acid, were also identified. Overall, these previously unreported forms constitute a substantial amount of fpB (up to approximately 17% of the total), and should be taken into account for a reliable quantitative analysis of fpB release.

Use of a double resonance electron capture dissociation experiment to probe fragment intermediate lifetimes.

The relative abundances of fragment ions in electron capture dissociation (ECD) are often greatly affected by the secondary and tertiary structures of the precursor ion, and have been used to derive the gas-phase conformations of the protein ions. In this study, it is found that resonance ejection of the charge reduced molecular ion during ECD resulted in significant changes in many fragment ion populations. The ratio of the ion peak intensities in the double resonance (DR)-ECD to that in the normal ECD experiment can be used to calculate the lifetime of the radical intermediates from which these fragments are derived. These lifetimes are often in the ms range, a time sufficiently long to facilitate multiple free radical rearrangements. These ratios correlate with the intramolecular noncovalent interactions in the precursor ion, and can be used to deduce information about the gas-phase conformation of peptide ions. DR-ECD experiments can also provide valuable information on ECD mechanisms, such as the importance of secondary electron capture and the origin of c./z ions.

The Effects of Trivalent Lanthanide Cationization on the Electron Transfer Dissociation of Acidic Fibrinopeptide B and its Analogs.

Electrospray ionization (ESI) on mixtures of acidic fibrinopeptide B and two peptide analogs with trivalent lanthanide salts generates [M + Met + H](4+), [M + Met](3+), and [M + Met -H](2+), where M = peptide and Met = metal (except radioactive promethium). These ions undergo extensive and highly efficient electron transfer dissociation (ETD) to form metallated and non-metallated c- and z-ions. All metal adducted product ions contain at least two acidic sites, which suggest attachment of the lanthanide cation at the side chains of one or more acidic residues. The three peptides undergo similar fragmentation. ETD on [M + Met + H](4+) leads to cleavage at every residue; the presence of both a metal ion and an extra proton is very effective in promoting sequence-informative fragmentation. Backbone dissociation of [M + Met](3+) is also extensive, although cleavage does not always occur between adjacent glutamic acid residues. For [M + Met - H ](2+), a more limited range of product ions form. All lanthanide metal peptide complexes display similar fragmentation except for europium (Eu). ETD on [M + Eu - H](2+) and [M + Eu](3+) yields a limited amount of peptide backbone cleavage; however, [M + Eu + H](4+) dissociates extensively with cleavage at every residue. With the exception of the results for Eu(III), metallated peptide ion formation by ESI, ETD fragmentation efficiencies, and product ion formation are unaffected by the identity of the lanthanide cation. Adduction with trivalent lanthanide metal ions is a promising tool for sequence analysis of acidic peptides by ETD. Graphical Abstract ᅟ.

Functional analysis of recombinant Bbeta15C and Bbeta15A fibrinogens demonstrates that Bbeta15G residue plays important roles in FPB release and in lateral aggregation of protofibrils.

BACKGROUND AND OBJECTIVES: Analysis of dysfibrinogens has improved our understanding of molecular defects and their effects on the function of intact fibrinogen. To eliminate the influence of plasma heterozygous molecules, we synthesized and analyzed recombinant-variant fibrinogens. METHODS: We synthesized two recombinant-variant fibrinogens with a single amino acid substitution at the 15Gly residue in the Bbeta-chain: namely, Bbeta15Cys and Bbeta15Ala. RESULTS: Western blotting analysis of purified fibrinogen revealed the existence of a small amount of a dimeric form only for Bbeta15Cys fibrinogen. For Bbeta15Cys fibrinogen, functional analysis indicated (a) no thrombin-catalyzed fibrinopeptide B (FPB) release and (b) markedly impaired lateral aggregation in thrombin- and reptilase-catalyzed fibrin polymerizations. For Bbeta15Ala fibrinogen, such analysis indicated slight impairments of both thrombin-catalyzed FPB release and lateral aggregation in thrombin-catalyzed fibrin polymerization, but nearly normal lateral aggregation in reptilase-catalyzed fibrin polymerization. These impaired lateral aggregations were accompanied by thinner fibrin fiber diameters (determined by scanning electron microscopy of the corresponding fibrin clots). CONCLUSION: We conclude that a region adjacent to Bbeta15Gly plays important roles in lateral aggregation not only in desA fibrin polymerization, but also in desAB fibrin polymerization, and we speculate that the marked functional differences between Bbeta15A and Bbeta15C fibrinogens in FPB release and fibrin polymerization might not only be due to the presence of a substituted cysteine residue in Bbeta15C fibrinogen, but also to the existence of disulfide-bonded forms. Finally, our data indicate that the Bbeta15Gly residue plays important roles in FPB release and lateral aggregation of protofibrils.

Electrospray ionisation mass spectrometry facilitates detection of fibrinogen (Bbeta 14 Arg --> Cys) mutation in a family with thrombosis.

We report the first direct detection of a fibrinogen mutation by electrospray ionisation mass spectrometry. The propositus, from a family with a history of thrombosis, came to attention after a pulmonary embolism subsequent to a spontaneous abortion. Prolonged thrombin (41 s) and reptilase times (26 s) together with an impairment of fibrinopeptide B release suggested a mutation at the thrombin cleavage site of the Bbeta chain. Direct mass analysis of purified fibrin chains from a thrombin induced clot showed that 50% of the Bbeta chains remained uncleaved. The measured mass of the mono sialo isoform of this uncleaved chain was 54150 Da, compared to a value of 54198 Da for normal Bbeta chains. This decrease of 48 Da in the intact protein is indicative of either a Bbeta 14 Arg to Cys, or Arg to Leu substitution. Heterozygosity for the Bbeta 14 Arg --> Cys mutation was verified by PCR amplification and DNA sequence analysis.

Negative ion matrix-assisted laser desorption/ionization time-of-flight post-source decay calibration by using fibrinopeptide B.

Fibrinopeptide B (M(r) 1552.58) was employed as a calibration compound for matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) post-source decay (PSD) fragment ion analysis in the negative mode. Experiments were performed by using both continuous and delayed extraction, with the maximum reflectron voltages being 30 and 21 kV, respectively. For comparison, a common positive ion PSD calibrant, ACTH(18-39) (M(r) 2466.7), was also employed with positive ion calibration constants being applied to negative ion spectra. Using fibrinopeptide B as the calibrant, the negative ion PSD results for angiotensin II (M(r) 1046.2), renin substrate tetradecapeptide (horse) (M(r) 1759.0), and the custom-synthesized peptide (K2G4)2 (M(r) 987.1) showed a factor of 1.5-2 improvement in absolute mass accuracy. Typical absolute mass-to-charge ratio accuracies were within +/- 1 Thompson and were achieved even when the peptide being analyzed was more massive than fibrinopeptide B. In addition, both calibrants showed increased accuracy when experiments were conducted in the delayed extraction mode. Other advantages of using fibrinopeptide B are its moderate cost and the ability to perform calibration and sample analysis for negative ion PSD under the same instrumental conditions.

Dissociation of multiply charged negative ions for hirudin (54-65), fibrinopeptide B, and insulin A (oxidized).

Collision-induced dissociation (CID) was performed on multiply deprotonated ions from three commercial peptides: hirudin (54-65), fibrinopeptide B, and oxidized insulin chain A. Ions were produced by electrospray ionization in a Fourier transform ion cyclotron resonance mass spectrometer. Each of these peptides contains multiple acidic residues, which makes them very difficult to ionize in the positive mode. However, the peptides deprotonate readily making negative ion studies a viable alternative. The CID spectra indicated that the likely deprotonation sites are acidic residues (aspartic, glutamic, and cysteic acids) and the C-terminus. The spectra are rife with c, y, and internal ions, although some a, b, x, and z ions form. Many of the fragment ions were formed from cleavage adjacent to acidic residues, both N- and C-terminal to the acidic site. In addition, neutral loss (e.g., NH3, CH3, H2O, and CO2) was prevalent from both the parent ions and from fragment ions. These neutral eliminations were often indicative of specific amino acid residues. The fragmentation patterns from several charge states of the parent ions, when combined, provide significant primary sequence information. These results suggest that negative mode CID of multiply deprotonated ions provides useful structural information and can be worthwhile for highly acidic peptides that do not form positive ions in abundance.

Endothelial desquamating activity of rat synthetic fibrinopeptide B and its analogues "in vivo": identification of responsible sequence.

The endothelial desquamating activity of the synthetic rat fibrinopeptide B (ATTDSDKVDLSIAR-OH), and its analogues was studied "in vivo" after intravenous administration to rats. Rat fibrinopeptide B (FPB) caused a significant increase in the count of circulating endothelial cell carcasses at the dose of 100 nmol/kg. Maximal effect reaching about 270% of the normal value was achieved with the dose of 600 nmol/kg in 30 min. after the injection. No significant thrombocytopenia, no hemolysis and no other life-threatening complications were observed. The same endothelial desquamating effect was achieved with N-terminal FPB(1-7) peptide ATTDSDK-OH, but very low activity exhibited the two FPB mutant peptides: ATDSDKVDLSIAR-OH and ATTNSNK-OH. Our results indicate that N-terminal sequence (1-7) consisting of N-terminal "pig tail" (ATT), acid region (DSD) and basic amino acid (K) is responsible for endothelial desquamating activity of rat FPB. Similar corresponding sequences may be recognized in FPB of different species. The conservation of this common "active site" sequence is less apparent in primates.

Generation and partial characterization of five monoclonal antibodies with high affinities for fibrin.

Five fibrin-specific monoclonal antibodies have been generated using three different immunogens and a unique clot/plasma screening procedure. Two of the mAbs are directed to an epitope(s) on the A alpha-chain of fibrinogen while two others seem to react with a distinct B beta-chain epitope(s). A third antibody seems to displace all the mAbs from fibrin and may be directed to a repeating structural domain in the fibrin. The Kd values obtained (approximately 10(-9)M) using dried fibrin clots suggest high avidity for fibrin. All the mAbs cross-react with the fibrin-like X-oligomer structure in solution but in the presence of fibrin this reaction seems irrelevant due to the low Kd of the mAb/fibrin reaction. These mAbs have been examined for uptake by fibrin in a laboratory circulation circuit and while the uptake was dependent on mAb concentration an increase was noted for most concentrations over the first 2 h of circulation in the presence of plasma.

Structural difference between polymerized and non-polymerized fragment X, obtained by plasmin digest of fibrinogen.

Fragment X (LMrFX) was obtained as low molecular weight preparations from a late stage 2 plasmin digest of human fibrinogen. The thrombin-treated LMrFX preparations, which resulted in impaired polymerization, were further subfractionated into polymerized and non-polymerized components. The fractions were examined by SDS-PAGE and immunochemical methods. In polymerized fractions, more peptide bands were observed on SDS-PAGE in the reduced state than in non-polymerized fractions. Both fractions contained a similar number of internal cleavages in the A alpha, B beta and gamma chains, which are linked by disulfide bonds. Thus, the partial deficiencies in polymerization sites of the carboxy terminal region of the gamma chain and the amino terminal portions of the B beta chain, as well as internal cleavage, were considered to participate in the impairment of the thrombin-induced polymerization of LMrFX.

C-terminal protein characterization by mass spectrometry using combined micro scale liquid and solid-phase derivatization.

A sample preparation method for protein C-terminal peptide isolation has been developed. In this strategy, protein carboxylate glycinamidation was preceded by carboxyamidomethylation and optional α- and ϵ-amine acetylation in a one-pot reaction, followed by tryptic digestion of the modified protein. The digest was adsorbed on ZipTip(C18) pipette tips for sequential peptide α- and ϵ-amine acetylation and 1-ethyl-(3-dimethylaminopropyl) carbodiimide-mediated carboxylate condensation with ethylenediamine. Amino group-functionalized peptides were scavenged on N-hydroxysuccinimide-activated agarose, leaving the C-terminal peptide in the flow-through fraction. The use of reversed-phase supports as a venue for peptide derivatization enabled facile optimization of the individual reaction steps for throughput and completeness of reaction. Reagents were exchanged directly on the support, eliminating sample transfer between the reaction steps. By this sequence of solid-phase reactions, the C-terminal peptide could be uniquely recognized in mass spectra of unfractionated digests of moderate complexity. The use of the sample preparation method was demonstrated with low-level amounts of a model protein. The C-terminal peptides were selectively retrieved from the affinity support and proved highly suitable for structural characterization by collisionally induced dissociation. The sample preparation method provides for robustness and simplicity of operation using standard equipment readily available in most biological laboratories and is expected to be readily expanded to gel-separated proteins.