Selective collision-induced fragmentation of ortho-hydroxybenzyl-aminated lysyl-containing tryptic peptides.

RATIONALE: In protein studies that employ tandem mass spectrometry the manipulation of protonated peptide fragmentation through exclusive dissociation pathways may be preferred in some applications over the comprehensive amide backbone fragmentation that is typically observed. In this study, we characterized the selective cleavage of the side-chain Cζ-Nε bond of peptides with ortho-hydroxybenzyl-aminated lysine residues. METHODS: Internal lysyl residues of representative peptides were derivatized via reductive amination with ortho-hydroxybenzaldehyde. The modified peptides were analyzed using collision-induced dissociation (CID) on an Orbitrap tandem mass spectrometer. Theoretical calculations using computational methods (density functional theory) were performed to investigate the potential dissociation mechanisms for the Cζ-Nε bond of the derivatized lysyl residue resulting in the formation of the observed product ions. RESULTS: Tandem mass spectra of the derivatized peptide ions exhibit product peaks corresponding to selective cleavage of the side-chain Cζ-Nε bond that links the derivative to lysine. The ortho-hydroxybenzyl derivative is released either as a neutral moiety [C7H6O1] or as a carbocation [C7H7O1](+) through competing pathways (retro-Michael versus Carbocation Elimination (CCE), respectively). The calculated transition state activation barriers indicate that the retro-Michael pathway is kinetically favored over CCE and both are favored over amide cleavage. CONCLUSIONS: The application of ortho-hydroxybenzyl amination is a promising peptide derivatization scheme for promoting selective dissociation pathways in the tandem mass spectrometry of protonated peptides. This can be implemented in the rational development of peptide reactive reagents for applications that may benefit from selective fragmentation paths (including crosslinking or MRM reagents).

Substituent effects on the gas-phase fragmentation reactions of protonated peptides containing benzylamine-derivatized lysyl residues.

Motivated by the need for chemical strategies designed to tune peptide fragmentation to selective cleavage reactions, benzyl ring substituent influence on the relative formation of carbocation elimination (CCE) products from peptides with benzylamine-derivatized lysyl residues has been examined using collision-induced dissociation (CID) tandem mass spectrometry. Unsubstituted benzylamine-derivatized peptides yield a mixture of products derived from amide backbone cleavage and CCE. The latter involves side-chain cleavage of the derivatized lysyl residue to form a benzylic carbocation [C(7)H(7)](+) and an intact peptide product ion [(MH(n))(n+) - (C(7)H(7))(+)]((n-1)+). The CCE pathway is contingent upon protonation of the secondary ε-amino group (N(ε)) of the derivatized lysyl residue. Using the Hammett methodology to evaluate the electronic contributions of benzyl ring substituents on chemical reactivity, a direct correlation was observed between changes in the CCE product ion intensity ratios (relative to backbone fragmentation) and the Hammett substituent constants, σ, of the corresponding substituents. There was no correlation between the substituent-influenced gas-phase proton affinity of N(ε) and the relative ratios of CCE product ions. However, a strong correlation was observed between the π orbital interaction energies (ΔE(int)) of the eliminated benzylic carbocation and the logarithm of the relative ratios, indicating the predominant factor in the CCE pathway is the substituent effect on the level of hyperconjugation and resonance stability of the eliminated benzylic carbocation. This work effectively demonstrates the applicability of σ (and ΔE(int)) as substituent selection parameters for the design of benzyl-based peptide-reactive reagents which tune CCE product formation as desired for specific applications.

Identification of novel Escherichia coli ribosome-associated proteins using isobaric tags and multidimensional protein identification techniques.

Biogenesis of the large ribosomal subunit requires the coordinate assembly of two rRNAs and 33 ribosomal proteins. In vivo, additional ribosome assembly factors, such as helicases, GTPases, pseudouridine synthetases, and methyltransferases, are also critical for ribosome assembly. To identify novel ribosome-associated proteins, we used a proteomic approach (isotope tagging for relative and absolute quantitation) that allows for semiquantitation of proteins from complex protein mixtures. Ribosomal subunits were separated by sucrose density centrifugation, and the relevant fractions were pooled and analyzed. The utility and reproducibility of the technique were validated via a double duplex labeling method. Next, we examined proteins from 30S, 50S, and translating ribosomes isolated at both 16 degrees C and 37 degrees C. We show that the use of isobaric tags to quantify proteins from these particles is an excellent predictor of the particles with which the proteins associate. Moreover, in addition to bona fide ribosomal proteins, additional proteins that comigrated with different ribosomal particles were detected, including both known ribosomal assembly factors and unknown proteins. The ribosome association of several of these proteins, as well as others predicted to be associated with ribosomes, was verified by immunoblotting. Curiously, deletion mutants for the majority of these ribosome-associated proteins had little effect on cell growth or on the polyribosome profiles.

ProteomeCommons.org IO Framework: reading and writing multiple proteomics data formats.

MOTIVATION: Effective use of proteomics data, specifically mass spectrometry data, relies on the ability to read and write the many mass spectrometer file formats. Even with mass spectrometer vendor-specific libraries and vendor-neutral file formats, such as mzXML and mzData it can be difficult to extract raw data files in a form suitable for batch processing and basic research. Introduced here are the ProteomeCommons.org Input and Output Framework, abbreviated to IO Framework, which is designed to abstractly represent mass spectrometry data. This project is a public, open-source, free-to-use framework that supports most of the mass spectrometry data formats, including current formats, legacy formats and proprietary formats that require a vendor-specific library in order to operate. The IO Framework includes an on-line tool for non-programmers and a set of libraries that developers may use to convert between various proteomics file formats. AVAILABILITY: The current source-code and documentation for the ProteomeCommons.org IO Framework is freely available at http://www.proteomecommons.org/current/531/

ProteomeCommons.org JAF: reference information and tools for proteomics.

SUMMARY: Analysis of proteomics data, specifically mass spectrometry data, commonly relies on libraries of known information such as atomic masses, known stable isotopes, atomic compositions of amino acids, observed modifications of known amino acids and ion masses that directly correspond to known amino acid sequences. The Java Analysis Framework (JAF) for proteomics provides a freely usable, open-source library of Java code that abstracts all of the aforementioned data, enabling more rapid development of proteomics tools. The JAF also includes several user tools that can be run directly from a web browser. AVAILABILITY: The current version and an archive of all older versions of the Java Analysis Framework for Proteomics is freely available, including complete source-code, at http://www.proteomecommons.org/current/511/.

Analysis of the outer membrane proteome of Caulobacter crescentus by two-dimensional electrophoresis and mass spectrometry.

Caulobacter crescentus, a Gram negative alpha-purple bacterium that displays an invariant asymmetric cell division pattern, has become a key model system for the study of bacterial development. Membrane proteins play key roles in cell cycle events, both as components of landmark morphological structures and as critical elements in regulation of the cell cycle. Recent advances for the isolation and solubilization of bacterial membrane proteins prior to isoelectric focusing have significantly improved the separation of outer membrane proteins by two-dimensional (2-D) electrophoresis. In this work we describe the analysis of the outer membrane proteome of Caulobacter crescentus. Proteins were identified using 2-D gel electrophoresis and peptide mass fingerprinting by matrix-assisted laser desorption/ionization-time of flight mass spectrometry. We identified 54 unique proteins out of which 41 were outer membrane proteins. Of the outer membrane proteins, 16 were identified as TonB-dependent receptor proteins. These studies were executed simultaneously with the Caulobacter genome sequencing project and advantages and limitations of proteomic analysis of a nonannotated genome are discussed. Finally, protein levels between cells grown in rich and minimal media are compared which demonstrates that many of the TonB-dependent receptor proteins are found at higher levels in minimal medium.

Virtual 2-D gel electrophoresis: visualization and analysis of the E. coli proteome by mass spectrometry.

Mass spectrometric surface analysis of isoelectric focusing gels provides an ultrasensitive approach to proteome analysis. This "virtual 2-D gel" approach, in which mass spectrometry is substituted for the size-based separation of SDS-PAGE, provides advantages in mass resolution and accuracy over classical 2-D gels and can be readily automated. Protein identities can be postulated from molecular mass (+/-0.1-0.2% for proteins of <50 kDa in size) and pI (+/-0.3 pH unit) and confirmed by MALDI in-source decay of the intact protein (providing sequence spanning up to 43 residues) or by peptide mass mapping following gel-wide chemical cleavage. Additionally, posttranslational modifications such as fatty acid acylation can be detected by the mass-resolved heterogeneity of component hydrocarbon chains. Sensitivity was evaluated by comparing the number of proteins detected by this method to equivalently loaded silver-stained 2-D gels. In the 5.7-6.0 pH range, E. coli is predicted to contain 435 proteins; virtual 2-D gels found 250 proteins ranging from >2 to <120 kDa in size present at levels to tens of femtomoles, as compared to the 100 proteins found by silver-staining 2-D gels. Extrapolating this result to the total theoretical proteome suggests that this technology is capable of detecting over 2500 E. coli proteins.

Two-dimensional electrophoresis and peptide mass fingerprinting of bacterial outer membrane proteins.

Many bacterial outer membrane proteins (OMPs) are missing from two-dimensional (2-D) gel proteome maps. Recently, we developed a technique for 2-D electrophoresis (2-DE) of Escherichia coli OMPs using alkaline pH incubation for isolation of OMPs, followed by improved solubilization conditions for array by 2-DE using immobilized pH gradients. In this report, we expanded our study, examining protein components from the outer membranes of two enteric bacteria, Salmonella typhimurium and Klebsiella pneumoniae (also known as Klebsiella aerogenes), as well as the unrelated, free-living alpha-proteobacteria Caulobacter crescentus. Patterns of OMPs expression appeared remarkably conserved between members of the Enterobacteriaceae, while C. crescentus was unique, displaying a greater number of clusters of higher-molecular-weight proteins (>80 kDa). Peptide mass fingerprinting (PMF) was used for protein identification, and despite matching across-species boundaries, proved useful for first-pass protein assignment of enteric OMPs. In contrast, identification of C. crescentus OMPs was successful only when searching against its recently completed genome. For all three microorganisms examined, the majority of proteins identified on the 2-D gel appear localized to the outer membrane, a result consistent with our previous finding in Escherichia coli. In addition, we discuss some of the benefits and limitations of PMF in cross-species searching.

Mass spectrometric imaging of immobilized pH gradient gels and creation of "virtual" two-dimensional gels.

We have developed a matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) based technique for the detection of intact proteins directly from immobilized pH gradient gels (IPGs). The use of this technique to visualize proteins from IPGs was explored in this study. Whole cell Escherichia coli extracts of various loadings were separated on IPGs. These IPGs were processed to remove contaminants and to achieve matrix/analyte cocrystallization on the surface of the gel. Mass spectra were acquired by scanning the surface of the gel and were assimilated into a "virtual" two dimensional (2-D) gel. This virtual 2-D gel is analogous to a "classical" 2-D gel, except that the molecular weight information is acquired by mass spectrometry rather than by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). This mass spectrometry (MS) based technology exemplifies a number of desirable characteristics, some of which are not attainable with classical two-dimensional electrophoresis (2-DE). These include high sensitivity, high reproducibility, and an inherently higher resolution and mass accuracy than 2-D gels. Furthermore, there is a difference in selectivity exhibited between virtual 2-D gels and classical 2-D gels, as a number of proteins are visible in the virtual gel image that are not present in the stained gels and vice versa. In this report, virtual 2-D gels will be compared to classical 2-D gels to illustrate these features.

HSP22, a new member of the small heat shock protein superfamily, interacts with mimic of phosphorylated HSP27 ((3D)HSP27).

Most of the members of the superfamily of mammalian small heat shock or stress proteins are abundant in muscles where they play a role in muscle function and maintenance of muscle integrity. One member of this protein superfamily, human HSP27, is rapidly phosphorylated on three serine residues (Ser(15), Ser(78), and Ser(82)) during cellular response to a number of extracellular factors. To understand better the role of HSP27, we performed a yeast two-hybrid screen of a human heart cDNA library for HSP27-interacting proteins. By using the triple aspartate mutant, a mimic of phosphorylated HSP27, as "bait" construct, a protein with a molecular mass of 21.6 kDa was identified as an HSP27-binding protein. Sequence analysis revealed that this new protein shares an overall sequence identity of 33% with human HSP27. This protein also contains the alpha-crystallin domain in its C-terminal half, a hallmark of the superfamily of small stress proteins. Thus, the new protein itself is a member of this protein superfamily, and consequently we designated it HSP22. According to the two-hybrid data, HSP22 interacts preferentially with the triple aspartate form of HSP27 as compared with wild-type HSP27. HSP22 is expressed predominantly in muscles. In vitro, HSP22 is phosphorylated by protein kinase C (at residues Ser(14) and Thr(63)) and by p44 mitogen-activated protein kinase (at residues Ser(27) and Thr(87)) but not by MAPKAPK-2.

Phosphopeptide derivatization signatures to identify serine and threonine phosphorylated peptides by mass spectrometry.

The development of rapid, global methods for monitoring states of protein phosphorylation would provide greater insight for understanding many fundamental biological processes. Current best practices use mass spectrometry (MS) to profile digests of purified proteins for evidence of phosphorylation. However, this approach is beset by inherent difficulties in both identifying phosphopeptides from within a complex mixture containing many other unmodified peptides and ionizing phosphopeptides in positive-ion MS. We have modified an approach that uses barium hydroxide to rapidly eliminate the phosphoryl group of serine and threonine modified amino acids, creating dehydroamino acids that are susceptible to nucleophilic derivatization. By derivatizing a protein digest with a mixture of two different alkanethiols, phosphopeptide-specific derivatives were readily distinguished by MS due to their characteristic ion-pair signature. The resulting tagged ion pairs accommodate simple and rapid screening for phosphopeptides in a protein digest, obviating the use of isotopically labeled samples for qualitative phosphopeptide detection. MALDI-MS is used in a first pass manner to detect derivatized phosphopeptides, while the remaining sample is available for tandem MS to reveal the site of derivatization and, thus, phosphorylation. We demonstrated the technique by identifying phosphopeptides from beta-casein and ovalbumin. The approach was further used to examine in vitro phosphorylation of recombinant human HSP22 by protein kinase C, revealing phosphorylation of Thr-63.

In pursuit of ceruminolytic agents: a study of earwax composition.

OBJECTIVE: To assess the protein and simple sugar content of earwax in pursuit of better ceruminolytic agents. STUDY DESIGN: Collected earwax specimens were tested in several media for dissolution before being analyzed for amino acid and carbohydrate content. PATIENTS: The samples were obtained from eight random patients requiring ear plug removal. RESULTS: The amino acid composition differs considerably from hair and stratum corneum of glabrous skin. Sugar analysis revealed high levels of galactosamine and galactose. CONCLUSIONS: This analysis of proteins and carbohydrates further characterizes earwax. Future ceruminolytic agents must dissolve lipid, keratin, and a monolayer of lipid covalently bound to the epidermal cells, which encourages cellular aggregation.

High sensitivity mass spectrometric methods for obtaining intact molecular weights from gel-separated proteins.

The molecular weight measurement of intact Escherichia coli proteins separated by isoelectric focusing-immobilized pH gradient (IEF-IPG) gels and analyzed by mass spectrometry is presented. Two methods are discussed: (i) electrospray ionization (ESI) mass spectrometry (MS) of extracted proteins, and (ii) matrix-assisted laser desorption/ionization (MALDI)-MS analysis directly from IEF-IPG gels. Both ESI and MALDI methods yield sub-picomole sensitivity and good mass measurement accuracy. The use of an array detector for ESI-MS was essential to discriminate against contaminating background ions and to selectively detect high mass protein ions. MALDI-MS offers high-throughput analysis of one- and potentially two-dimensional (2-D) gels. The "virtual 2-D" gel method with first-dimensional IEF separation and the second dimension as molecular mass determination by MS, is a particularly promising method for protein analysis due to its ultra high sensitivity and correspondence to classical 2-D gels. Further sensitivity enhancements for the MALDI-MS method are provided by post acceleration detection optimized for high mass time-of-flight analysis.

Determination of low-molecular-weight heparins and their binding to protamine and a protamine analog using polyion-sensitive membrane electrodes.

A polycation-sensitive membrane electrode based on the ion-exchanger dinonylnaphthalene sulfonate has previously been developed and used as an end-point detector for the determination of unfractionated heparin in whole blood samples via simple potentiometric titration with protamine. Herein, we report the application of the same methodology for the quantitation of a commercial low-molecular-weight heparin (LMWH) preparation (Fragmin) in whole blood samples at concentrations up to 2 U/ml. Further, an analogous polyanion (heparin)-sensitive electrode is used to estimate the binding constants between protamine and various LMWH preparations. The equilibrium constants (Keq) and the number of binding sites per mole of heparin (n) are determined by recasting the data in the form of a Scatchard plot. Results show that the average molecular weight and molecular weight distribution of the LMWH preparation are important parameters affecting their binding with protamine. Comparable binding constants are obtained for the same LMWH preparations titrated with a synthetic protamine analog, [+18RGD] [acetyl-EA(R2A2R2A)4R2GRGDSPA-NH2].

AP30, a differential protein marker for perilymph and cerebrospinal fluid in middle ear fluid, has been purified and identified as human apolipoprotein D.

Using two-dimensional (2-D) gel electrophoresis, human perilymph and cerebrospinal fluid have been shown to be highly enriched for an acidic protein with MR 30,000, we designated it as AP30. The protein exhibits charge heterogeneity, with at least eight isoforms visible between pI 4.5 to 5.5 on 2-D gels. Purification of the protein was carried out by ammonium sulfate precipitation, polybuffer exchanger column chromatofocusing, and acetone fractional precipitation. The resulting preparation also contains eight spots in the acidic area of 2-D gels, and one broad band located at Mr 30,000 by SDS-PAGE. Digestion of AP30 with neuraminidase causes the isoforms to shift to a more basic position and to consolidate into two primary spots, indicating that AP30 is a variably sialylated glycoprotein. Amino acid analysis of AP30 revealed an amino acid content very similar to that of human apolipoprotein D. Attempts to determine the amino acid sequence demonstrated that the N-terminus is blocked. Edman sequencing of two peptide fragments, generated by cyanogen bromide cleavage of AP30, both revealed sequences having 100% identity to human apolipoprotein D. Western blot analysis of AP30 with the antibody against authentic human apolipoprotein D demonstrated a high degree of cross-reactivity. These studies indicate that AP30 from human perilymph and cerebrospinal fluid is a member of the apolipoprotein D family.

Revised primary structures of rat pituitary gamma-lipotrophin and beta-endorphin.

In-depth investigations, by high performance liquid chromatographic purification, radio-immunoassay, mass spectrometry, tandem mass spectrometry, Edman sequencing and limited C-terminal ladder sequencing, were prompted by mass spectrometric charting experiments which suggested that the amino acid sequences for rat gamma-lipotrophin and beta-endorphin require revision. The results for gamma-lipotrophin identify a histidine for glutamine substitution at position 12, and heterogeneity in the expressed protein presumably due to partial dehydration. Partial dehydration for acidic joining peptide, previously reported by Toney et al was corroborated. The results for beta-endorphin confirm the presence of alanine at position 26 and provide no evidence for the expression of multiple forms of the hormone.

Comparison of the hemodynamic and hematologic toxicity of a protamine variant after reversal of low-molecular-weight heparin anticoagulation in a canine model.

Using the dog as an animal model, we developed an experimental preparation to compare hemodynamic and hematologic toxicity of anticoagulation reversal. Currently, protamine sulfate reversal of standard unfractionated heparin and low-molecular-weight heparin (LMWH) anticoagulation causes adverse side effects, including decreased systemic mean arterial pressure (MAP), decreased cardiac output (CO), decreased oxygen consumption (VO2), and thrombocytopenia. In addition, standard protamine is only marginally effective at reversing the factor Xa inhibition induced by LMWHs. We have produced protamine-like variant peptides to decrease the adverse responses attributed to standard protamine. The hemodynamic, hematologic, and coagulation effects of standard protamine and the protamine variant (+18RGD) were assessed after reversal of LMWH anticoagulation in anesthetized dogs. Flow probes and vascular catheters were surgically implanted for measurement of hemodynamic parameters including MAP, CO, VO2, and heart rate (HR). Hematologic studies (platelet and white blood cell counts) and coagulation studies (activated clotting time [ACT], activated partial thromboplastin time [aPTT], thrombin clotting time [TCT], antifactor Xa and antifactor IIa values) also were performed. The protamine variant +18RGD was less toxic, induced less thrombocytopenia, and was more effective in anticoagulation reversal than was standard protamine sulfate. Results of this study indicate that the dog may be a useful model for investigating important hemodynamic, hematologic, and coagulation parameters during reversal of LMWH anticoagulation by use of synthetic protamine variants.

Sensitivity and mass accuracy for proteins analyzed directly from polyacrylamide gels: implications for proteome mapping.

Matrix-assisted laser desorption ionization (MALDI) mass spectra have been obtained directly from thin-layer isoelectric focusing (IEF) gels with as little as 700 femtomoles of alpha- and beta-chain bovine hemoglobin and bovine carbonic anhydrase, and 2 picomoles of bovine trypsinogen, soybean trypsin inhibitor, and bovine serum albumin all loaded onto a single lane. By soaking the gel in a matrix solution, matrix was deposited over the entire gel surface, allowing MALDI scanning down complete lanes of the one-dimensional gel. As long as matrix crystals were deposited finely on the surface of the gel, time-lag focusing techniques were capable of ameliorating some of the mass accuracy limitations inherent in desorbing from uneven insulator surfaces with external calibration. Eleven measurements on the 5 kDa alpha-subunit proteins of lentil lectin measured over the course of 1 h and referenced to a single calibration yielded a standard deviation of 0.025%. Colloidal gold staining was found to be compatible with desorption directly from IEF and sodium dodecyl sulfate (SDS)-polyacrylamide gels. This direct approach simplifies the interface between gel electrophoresis and mass spectrometry dramatically, making the process more amenable to automation.

Differential effects of novel protamine variants on myocyte contractile function with left ventricular failure.

BACKGROUND: Protamine administration can cause left ventricular (LV) dysfunction, which may have clinical significance in the setting of congestive heart failure (CHF). Protamine variants have recently been constructed with heparin reversal capacity similar to protamine. The purpose of this study was to examine the potential differential effects of these protamine variants on isolated myocyte contractile function in normal myocytes and in myocytes after the development of CHF. METHODS: Contractile function was measured by means of computer-aided videomicroscopy in myocytes from five normal pigs and five pigs with CHF induced by rapid pacing (240 beats/min for 3 weeks). Myocyte contractility was examined in the presence of 40 micrograms/ml native protamine or one of three protamine variants: (1) reduced charge (+18) and lysine substituted for arginine; (2) lysine-substituted variant with glutamic acid substituted for the initial proline; or (3) arginine-rich peptide with a terminal arginine-glycine-aspartic acid (RGD) amino acid sequence. RESULTS: In the presence of native protamine, myocyte percent shortening fell from baseline in both the normal (2.86 +/- 0.15 versus 4.58 +/- 0.08, p < 0.05) and the CHF groups (1.01 +/- 0.06 versus 2.07 +/- 0.05, p < 0.05). With both of the lysine-substituted protamine variants, percent shortening fell from baseline in the normal group (3.42 +/- 0.20 for arginine and 3.74 +/- 0.20 for glutamic acid versus 4.58 +/- 0.08, p < 0.05), and was unchanged in the CHF group (1.94 +/- 0.13 versus 2.07 +/- 0.05, p = 0.34 for arginine; and 1.96 +/- 0.10 versus 2.07 +/- 0.05, p = 0.31, for glutamic acid). However, with the arginine/RGD variant, percent shortening fell from baseline in both the normal (2.86 +/- 0.23 versus 4.58 +/- 0.08, p < 0.05) and the CHF groups (1.32 +/- 0.10 versus 2.07 +/- 0.05, p < 0.05). CONCLUSIONS: Specific changes in the primary and secondary structures of protamine had different effects on myocyte contractile function. Furthermore, the negative effects of lysine-substituted protamine variants on myocyte contractility were less pronounced in both CHF and normal myocytes. Thus protamine variants may be of clinical use, particularly in the setting of preexisting LV dysfunction.