Mass spectrometric analysis of a UV-cross-linked protein-DNA complex: tryptophans 54 and 88 of E. coli SSB cross-link to DNA

Protein-nucleic acid complexes are commonly studied by photochemical cross-linking. UV-induced cross-linking of protein to nucleic acid may be followed by structural analysis of the conjugated protein to localize the cross-linked amino acids and thereby identify the nucleic acid binding site. Mass spectrometry is becoming increasingly popular for characterization of purified peptide-nucleic acid heteroconjugates derived from UV cross-linked protein-nucleic acid complexes. The efficiency of mass spectrometry-based methods is, however, hampered by the contrasting physico-chemical properties of nucleic acid and peptide entities present in such heteroconjugates. Sample preparation of the peptide-nucleic acid heteroconjugates is, therefore, a crucial step in any mass spectrometry-based analytical procedure. This study demonstrates the performance of four different MS-based strategies to characterize E. coli single-stranded DNA binding protein (SSB) that was UV-cross-linked to a 5-iodouracil containing DNA oligomer. Two methods were optimized to circumvent the need for standard liquid chromatography and gel electrophoresis, thereby dramatically increasing the overall sensitivity of the analysis. Enzymatic degradation of protein and oligonucleotide was combined with miniaturized sample preparation methods for enrichment and desalting of cross-linked peptide-nucleic acid heteroconjugates from complex mixtures prior to mass spectrometric analysis. Detailed characterization of the peptidic component of two different peptide-DNA heteroconjugates was accomplished by matrix-assisted laser desorption/ionization mass spectrometry and allowed assignment of tryptophan-54 and tryptophan-88 as candidate cross-linked residues. Sequencing of those peptide-DNA heteroconjugates by nanoelectrospray quadrupole time-of-flight tandem mass spectrometry identified tryptophan-54 and tryptophan-88 as the sites of cross-linking. Although the UV-cross-linking yield of the protein-DNA complex did not exceed 15%, less than 100 pmole of SSB protein was required for detailed structural analysis by mass spectrometry.     

Identification of a cross-link in the Escherichia coli ribosomal protein pair S13-S19 at the amino acid level

Escherichia coli 30 S ribosomal subunits and 70 S ribosomes were treated with the bifunctional reagent diepoxybutane, acting as a cross-linker. One major cross-linked protein pair in the 30 S subunit was generated in relatively high yields. This cross-link was shown to consist of ribosomal proteins S13 and S19. Purification of this complex was achieved by a series of conventional and/or high pressure liquid chromatography techniques allowing its isolation in milligram quantities. To reveal the exact position of the two amino acids involved in the cross-link formation, the purified protein pair S13-S19 was subjected to several enzymatic fragmentations, and the resulting peptides were characterized by sequence analysis, amino acid analysis, and fast atom bombardment mass spectrometry. After isolation of the cross-linked peptides, Cys84 in protein S13 and His68 in S19 could be unequivocally identified as the amino acids cross-linked by the bifunctional reagent. This result demonstrates that, despite neutron scattering data which place the centers of mass of S13 and S19 85 A apart, at least these regions of the two proteins are located within a 4-A distance in the ribosomal particle.     

Photo-assisted peptide enrichment in protein complex cross-linking analysis of a model homodimeric protein using mass spectrometry

MS analysis of cross-linked peptides can be used to probe protein contact sites in macromolecular complexes. We have developed a photo-cleavable cross-linker that enhances peptide enrichment, improving the signal-to-noise ratio of the cross-linked peptides in mass spectrometry analysis. This cross-linker utilizes nitro-benzyl alcohol group that can be cleaved by UV irradiation and is stable during the multiple washing steps used for peptide enrichment. The enrichment method utilizes a cross-linker that aids in eliminating contamination resulting from protein-based retrieval systems, and thus, facilitates the identification of cross-linked peptides. Homodimeric pilM protein from Pseudomonas aeruginosa 2192 (pilM) was investigated to test the specificity and experimental conditions. As predicted, the known pair of lysine side chains within 14?? was cross-linked. An unexpected cross-link involving the protein's amino terminus was also detected. This is consistent with the predicted mobility of the amino terminus that may bring the amino groups within 19?? of one another in solution. These technical improvements allow this method to be used for investigating protein-protein interactions in complex biological samples.     

Sites of nucleic acid binding in type I-IV intermediate filament subunit proteins

A combination of enzymatic and chemical ladder sequencing of photo-cross-linked protein-single-stranded oligodeoxyribonucleotide complexes and analysis by MALDI-TOF mass spectrometry was employed to identify the amino acid residues responsible for the stable binding of nucleic acids in several intermediate filament (IF) subunit proteins. The IF proteins studied included the type I and type II cytokeratins K8, K18, and K19; the type III proteins desmin, glial fibrillary acidic protein (GFAP), peripherin, and vimentin; and the type IV neurofilament triplet protein L (NF-L). The site of nucleic acid binding was localized to the non-alpha-helical, amino-terminal head domain of all of the IF proteins tested. GFAP, which has the shortest head domain of the proteins tested, cross-linked via only two amino acid residues. One of these residues was located within a conserved nonapeptide domain that has been shown to be required for filament formation. One or more cross-linked residues were found in a similar location in the other proteins studied. The major binding site for nucleic acids for most of the proteins appears to be localized within the middle of the head domain. The two exceptions to this generalization are GFAP, which lacks these residues, and NF-L, in which a large number of cross-linked residues were found scattered throughout the first half of the head domain. Control experiments were also done with two bacteriophage ssDNA-binding proteins, as well as actin and tubulin. The single sites of cross-linkage observed with the bacteriophage proteins, Phe(183) for the T4 gene 32 protein and Phe(73) for the M13 gene 5 protein, were in good agreement with literature data. Actin and tubulin could not be cross-linked to the oligonucleotide. Aside from the insight into the biological activity of IF proteins that these data provide, they also demonstrate that this analytical method can be employed to study a variety of protein-nucleic acid interactions.     

Chemical cross-linking, mass spectrometry, and in silico modeling of proteasomal 20S core particles of the haloarchaeon Haloferax volcanii

A fast and accurate method is reported to generate distance constraints between juxtaposited amino acids and to validate molecular models of halophilic protein complexes. Proteasomal 20S core particles (CPs) from the haloarchaeon Haloferax volcanii were used to investigate the quaternary structure of halophilic proteins based on their symmetrical, yet distinct subunit composition. Proteasomal CPs are cylindrical barrel-like structures of four-stacked homoheptameric rings of α- and β-type subunits organized in α(7)β(7) β(7)α(7) stoichiometry. The CPs of H. volcanii are formed from a single type of β subunit associated with α1 and/or α2 subunits. Tandem affinity chromatography and new genetic constructs were used to separately isolate α1(7)β(7)β(7)α1(7) and α2(7)β(7)β(7)α2(7) CPs from H. volcanii. Chemically cross-linked peptides of the H. volcanii CPs were analyzed by high-performance mass spectrometry and an open modification search strategy to first generate and then to interpret the resulting tandem mass spectrometric data. Distance constraints obtained by chemical cross-linking mass spectrometry, together with the available structural data of nonhalophilic CPs, facilitated the selection of accurate models of H. volcanii proteasomal CPs composed of α1-, α2-, and β-homoheptameric rings from several different possible structures from Protein Data Bank.     

Identification of tyrosine 204 as the photo-cross-linking site in the DNA-EcoRI DNA methyltransferase complex by electrospray ionization mass spectrometry

We describe a highly sensitive strategy combining laser-induced photo-cross-linking and HPLC-based electrospray ionization mass spectrometry to identify amino acid residues involved in protein-DNA recognition. The photoactivatible cross-linking thymine isostere, 5-iodoracil, was incorporated at a single site within the sequence recognized by EcoRI DNA methyltransferase (GAATTC). UV irradiation of the DNA-protein complex at 313 nm results in a >60% cross-linking yield. SDS-polyacrylamide gel electrophoresis and mass spectrometry were used to analyze the covalent cross-linked complex. The total mass is consistent with covalent bond formation between one strand of DNA and the protein with 1:1 stoichiometry. Protease digestion of the cross-linked complex yields several peptide-DNA adducts that were purified by anion-exchange column chromatography. A combination of mass spectrometric analysis and amino acid sequencing revealed that tyrosine 204 was cross-linked to the DNA. Electrospray mass spectrometric analysis of the peptide-nucleoside adduct confirmed this assignment. Tyrosine 204 resides in a peptide motif previously thought to be involved in AdoMet binding and methyl transfer. Thus, amino acids within loop segments but outside of "DNA binding" motifs can be critical to DNA recognition. Our method provides an accurate characterization of picomole quantities of DNA-protein complexes.     

Theoretical and experimental prospects for protein identification based solely on accurate mass measurement

We discuss the theoretical and experimental potential and limitations of protein identification by mass measurement of proteolytic peptides and database searching. For peptides differing in composition by one (or two or three) amino acids, a surprisingly high number turn out to have isomers: 10% (or 29% or 53%), considering the 20 common amino acids with equal relative abundance. Even if isomers differing by leucine/isoleucine are excluded, the latter numbers are 14% and 38%--those isomeric peptides cannot be distinguished based on mass alone, and tandem mass spectrometry and/or other additional constraints are needed. However, for nominally isobaric peptides, the mass accuracy and resolving power of broadband Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry theoretically and experimentally suffice to resolve virtually all peptide doublets differing by up to two amino acids--including the smallest mass difference of 3.4 mDa. We demonstrate experimental resolution of another pair of peptides differing by 11 mDa, even when present in a complex mixture of hundreds of other peptides.     

Sequence analysis of peptide mixtures by automated integration of Edman and mass spectrometric data.

A computer algorithm is described that utilizes both Edman and mass spectrometric data for simultaneous determination of the amino acid sequences of several peptides in a mixture. Gas phase sequencing of a peptide mixture results in a list of observed amino acids for each cycle of Edman degradation, which by itself may not be informative and typically requires reanalysis following additional chromatographic steps. Tandem mass spectrometry, on the other hand, has a proven ability to analyze sequences of peptides present in mixtures. However, mass spectrometric data may lack a complete set of sequence-defining fragment ions, so that more than one possible sequence may account for the observed fragment ions. A combination of the two types of data reduces the ambiguity inherent in each. The algorithm first utilizes the Edman data to determine all hypothetical sequences with a calculated mass equal to the observed mass of one of the peptides present in the mixture. These sequences are then assigned figures of merit according to how well each of them accounts for the fragment ions in the tandem mass spectrum of that peptide. The program was tested on tryptic and chymotryptic peptides from hen lysozyme, and the results are compared with those of another computer program that uses only mass spectral data for peptide sequencing. In order to assess the utility of this method the program is tested using simulated mixtures of varying complexity and tandem mass spectra of varying quality.     

Protein cross-linking analysis using mass spectrometry, isotope-coded cross-linkers, and integrated computational data processing

Distance constraints in proteins and protein complexes provide invaluable information for calculation of 3D structures, identification of protein binding partners and localization of protein-protein contact sites. We have developed an integrative approach to identify and characterize such sites through the analysis of proteolytic products derived from proteins chemically cross-linked by isotopically coded cross-linkers using LC-MALDI tandem mass spectrometry and computer software. This method is specifically tailored toward the rapid analysis of low microgram amounts of proteins or multimeric protein complexes cross-linked with nonlabeled and deuterium-labeled bis-NHS ester cross-linking reagents (both commercially available and readily synthesized). Through labeling with [18O]water solvent and LC-MALDI analysis, the method further allows the possible distinction between Type 0 and Type 1 or Type 2 modified peptides (monolinks and looplinks or cross-links), although such a distinction is more readily made from analysis of tandem mass spectrometry data. When applied to the bacterial Colicin E7 DNAse/Im7 heterodimeric protein complex, 23 cross-links were identified including six intersubunit cross-links, all between residues that are close in space when examined in the context of the X-ray structure of the heterodimer. In addition, cross-links were successfully identified in five single subunit proteins, beta-lactoglobulin, cytochrome c, lysozyme, myoglobin, and ribonuclease A, establishing the generality of the approach.     

Free amino acid quantification by LC-MS/MS using derivatization generated isotope-labelled standards

The further development of derivatizing reagents for plasma amino acid quantification by tandem mass spectrometry is described. The succinimide ester of 4-methylpiperazineacetic acid (MPAS), the iTRAQ reagent, was systematically modified to improve tandem mass spectrometer (MS/MS) product ion intensity. 4-Methylpiperazinebutyryl succinimide (MPBS) and dimethylaminobutyryl succinimide (DMABS) afforded one to two orders of magnitude greater MS/MS product ion signal intensity than the MPAS derivative for simple amino acids. CD(3) analogues of the modified derivatizing reagents were evaluated for preparation of amino acid isotope-labelled quantifying standards. Acceptable accuracy and precision was obtained with d(3)-DMABS as the amino acid standards derivatizing reagent. The product ion spectra of the DMABS amino acid derivatives are diagnostic for structural isomers including valine/norvaline, alanine/sarcosine and leucine/isoleucine. Improved analytical sensitivity and specificity afforded by these derivatives may help to establish liquid chromatography tandem mass spectrometry (LC-MS/MS) with derivatization generated isotope-labelled standards a viable alternative to amino acids analysers.     

Analyzing proteomes and protein function using graphical comparative analysis of tandem mass spectrometry results

Although generating large amounts of proteomic data using tandem mass spectrometry has become routine, there is currently no single set of comprehensive tools for the rigorous analysis of tandem mass spectrometry results given the large variety of possible experimental aims. Currently available applications are typically designed for displaying proteins and posttranslational modifications from the point of view of the mass spectrometrist and are not versatile enough to allow investigators to develop biological models of protein function, protein structure, or cell state. In addition, storage and dissemination of mass spectrometry-based proteomic data are problems facing the scientific community. To address these issues, we have developed a relational database model that efficiently stores and manages large amounts of tandem mass spectrometry results. We have developed an integrated suite of multifunctional analysis software for interpreting, comparing, and displaying these results. Our system, Bioinformatic Graphical Comparative Analysis Tools (BIGCAT), allows sophisticated analysis of tandem mass spectrometry results in a biologically intuitive format and provides a solution to many data storage and dissemination issues.     

A suboptimal algorithm for de novo peptide sequencing via tandem mass spectrometry.

Tandem mass spectrometry has emerged to be one of the most powerful high-throughput techniques for protein identification. Tandem mass spectrometry selects and fragments peptides of interest into N-terminal ions and C-terminal ions, and it measures the mass/charge ratios of these ions. The de novo peptide sequencing problem is to derive the peptide sequences from given tandem mass spectral data of k ion peaks without searching against protein databases. By transforming the spectral data into a matrix spectrum graph G = (V, E), where |V| = O(k(2)) and |E| = O(k(3)), we give the first polynomial time suboptimal algorithm that finds all the suboptimal solutions (peptides) in O(p|E|) time, where p is the number of solutions. The algorithm has been implemented and tested on experimental data. The program is available at http://hto-c.usc.edu:8000/msms/menu/denovo.htm.     

Spectral networks: a new approach to de novo discovery of protein sequences and posttranslational modifications

Significant technological advances have accelerated high-throughput proteomics to the automated generation of millions of tandem mass spectra on a daily basis. In such a setup, the desire for greater sequence coverage combines with standard experimental procedures to commonly yield multiple tandem mass spectra from overlapping peptides-typical observations include peptides differing by one or two terminal amino acids and spectra from modified and unmodified variants of the same peptides. In a departure from the traditional spectrum identification algorithms that analyze each tandem mass spectrum in isolation, spectral networks define a new computational approach that instead finds and simultaneously interprets sets of spectra from overlapping peptides. In shotgun protein sequencing, spectral networks capitalize on the redundant sequence information in the aligned spectra to deliver the longest and most accurate de novo sequences ever reported for ion trap data. Also, by combining spectra from multiple modified and unmodified variants of the same peptides, spectral networks are able to bypass the dominant guess/confirm approach to the identification of posttranslational modifications and alternatively discover modifications and highly modified peptides directly from experimental data. Open-source implementations of these algorithms may be downloaded from peptide.ucsd.edu.     

Measuring protein synthesis using metabolic ²H labeling, high-resolution mass spectrometry, and an algorithm

We recently developed a method for estimating protein dynamics in vivo with heavy water ((2)H(2)O) using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) [16], and we confirmed that (2)H labeling of many hepatic free amino acids rapidly equilibrated with body water. Although this is a reliable method, it required modest sample purification and necessitated the determination of tissue-specific amino acid labeling. Another approach for quantifying protein kinetics is to measure the (2)H enrichments of body water (precursor) and protein-bound amino acid or proteolytic peptide (product) and to estimate how many copies of deuterium are incorporated into a product. In the current study, we used nanospray linear trap Fourier transform ion cyclotron resonance mass spectrometry (LTQ FT-ICR MS) to simultaneously measure the isotopic enrichment of peptides and protein-bound amino acids. A mathematical algorithm was developed to aid the data processing. The most notable improvement centers on the fact that the precursor/product labeling ratio can be obtained by measuring the labeling of water and a protein (or peptide) of interest, thereby minimizing the need to measure the amino acid labeling. As a proof of principle, we demonstrate that this approach can detect the effect of nutritional status on albumin synthesis in rats given (2)H(2)O.     

Mapping of the 5'-2-deoxyribose-5-phosphate lyase active site in DNA polymerase beta by mass spectrometry

The mechanism of the 5'-2-deoxyribose-5-phosphate lyase reaction catalyzed by mammalian DNA beta-polymerase (beta-pol) was investigated using a cross-linking methodology in combination with mass spectrometric analyses. The approach included proteolysis of the covalently cross-linked protein-DNA complex with trypsin, followed by isolation, peptide mapping, and mass spectrometric and tandem mass spectrometric analyses. The 8-kDa domain of beta-pol was covalently cross-linked to a 5'-2-deoxyribose-5-phosphate-containing DNA substrate by sodium borohydride reduction. Using tandem mass spectrometry, the location of the DNA adduct on the 8-kDa domain was unequivocally determined to be at the Lys(72) residue. No additional amino acid residues were found as minor cross-linked species. These data allow assignment of Lys(72) as the sole Schiff base nucleophile in the 8-kDa domain of beta-pol. These results provide the first direct evidence in support of a catalytic mechanism involving nucleophilic attack by Lys(72) at the abasic site.     

Protein-rRNA binding features and their structural and functional implications in ribosomes as determined by cross-linking studies.

We have investigated protein-rRNA cross-links formed in 30S and 50S ribosomal subunits of Escherichia coli and Bacillus stearothermophilus at the molecular level using UV and 2-iminothiolane as cross-linking agents. We identified amino acids cross-linked to rRNA for 13 ribosomal proteins from these organisms, namely derived from S3, S4, S7, S14, S17, L2, L4, L6, L14, L27, L28, L29 and L36. Several other peptide stretches cross-linked to rRNA have been sequenced in which no direct cross-linked amino acid could be detected. The cross-linked amino acids are positioned within loop domains carrying RNA binding features such as conserved basic and aromatic residues. One of the cross-linked peptides in ribosomal protein S3 shows a common primary sequence motif--the KH motif--directly involved in interaction with rRNA, and the cross-linked amino acid in ribosomal protein L36 lies within the zinc finger-like motif of this protein. The cross-linked amino acids in ribosomal proteins S17 and L6 prove the proposed RNA interacting site derived from three-dimensional models. A comparison of our structural data with mutations in ribosomal proteins that lead to antibiotic resistance, and with those from protein-antibiotic cross-linking experiments, reveals functional implications for ribosomal proteins that interact with rRNA.     

Cross-linking measurements of the Potato leafroll virus reveal protein interaction topologies required for virion stability, aphid transmission, and virus-plant interactions

Protein interactions are critical determinants of insect transmission for viruses in the family Luteoviridae. Two luteovirid structural proteins, the capsid protein (CP) and the readthrough protein (RTP), contain multiple functional domains that regulate virus transmission. There is no structural information available for these economically important viruses. We used Protein Interaction Reporter (PIR) technology, a strategy that uses chemical cross-linking and high resolution mass spectrometry, to discover topological features of the Potato leafroll virus (PLRV) CP and RTP that are required for the diverse biological functions of PLRV virions. Four cross-linked sites were repeatedly detected, one linking CP monomers, two within the RTP, and one linking the RTP and CP. Virus mutants with triple amino acid deletions immediately adjacent to or encompassing the cross-linked sites were defective in virion stability, RTP incorporation into the capsid, and aphid transmission. Plants infected with a new, infectious PLRV mutant lacking 26 amino acids encompassing a cross-linked site in the RTP exhibited a delay in the appearance of systemic infection symptoms. PIR technology provided the first structural insights into luteoviruses which are crucially lacking and are involved in vector-virus and plant-virus interactions. These are the first cross-linking measurements on any infectious, insect-transmitted virus.     

Quantitative urine amino acid analysis using liquid chromatography tandem mass spectrometry and aTRAQ reagents

Ion-exchange chromatography (IEC) is the most widely used method for amino acid analysis in physiological fluids because it provides excellent separation and reproducibility, with minimal sample preparation. The disadvantage, however, is the long analysis time needed to chromatographically resolve all the amino acids. To overcome this limitation, we evaluated a novel liquid chromatography tandem mass spectrometry (LC-MS/MS) method, which utilizes aTRAQ reagents, for amino acid analysis in urine. aTRAQ reagents tag the primary and secondary amino groups of amino acids. Internal standards for each amino acid are also labeled with a modified aTRAQ tag and are used for quantification. Separation and identification of the amino acids is achieved by liquid chromatography tandem mass spectrometry using retention times and mass transitions, unique to each amino acid, as identifiers. The run time, injection-to-injection, is 25 min, with all amino acids eluting within the first 12 min. This method has a limit of quantification (LOQ) of 1 μmol/L, and is linear up to 1000 μmol/L for most amino acids. The Coefficient of Variation (CV) was less than 20% for all amino acids throughout the linear range. Method comparison demonstrated concordance between IEC and LC-MS/MS and clinical performance was assessed by analysis of samples from patients with known conditions affecting urinary amino acid excretion. Reference intervals established for this method were also concordant with reference intervals obtained with IEC. Overall, aTRAQ reagents used in conjunction with LC-MS/MS should be considered a comparable alternative to IEC. The most attractive features of this methodology are the decreased run time and increased specificity.