Intra-organ cell specific mitochondrial quantitative interactomics.

Almost every organ consists of many cell types, each with its unique functions. Proteomes of these cell types are thus unique too. But it is reasonable to assume that interactome (inter and intra molecular interactions of proteins) are also distinct since protein interactions are what ultimately carry out the function. Podocytes and tubules are two cell types within kidney with vastly different functions: podocytes envelop the blood vessels in the glomerulus and act as filters while tubules are located downstream of the glomerulus and are responsible for reabsorption of important nutrients. It has been long known that for tubules mitochondria plays an important role as they require a lot of energy to carry out their functions. In podocytes, however, it has been assumed that mitochondria might not matter as much in both normal physiology and pathology1. Here we have applied quantitative cross-linking mass spectrometry to compare mitochondrial interactomes of tubules and podocytes using a transgenic mitochondrial tagging strategy to immunoprecipitate cell-specific mitochondria directly from whole kidney. We have uncovered that mitochondrial proteomes of these cell types are quite similar, although still showing unique features that correspond to known functions, such as high energy production through TCA cycle in tubules and requirements for detoxification in podocytes which are on the frontline of filtration where they encounter toxic compounds and therefore, as a non-renewing cell type they must have ways to protect themselves from cellular toxicity. But we gained much deeper insight with the interactomics data. We were able to find pathways differentially regulated in podocytes and tubules based on changing cross-link levels and not just protein levels. Among these pathways are betaine metabolism, lysine degradation, and many others. We have also demonstrated how quantitative interactomics could be used to detect different activity levels of an enzyme even when protein abundances of it are the same between cell types. We have validated this finding with an orthogonal activity assay. Overall, this work presents a new view of mitochondrial biology for two important, but functionally distinct, cell types within the mouse kidney showing both similarities and unique features. This data can continue to be explored to find new aspects of mitochondrial biology, especially in podocytes, where mitochondria has been understudied. In the future this methodology can also be applied to other organs to uncover differences in the function of cell types.

Role of the tumor suppressor IQGAP2 in metabolic homeostasis: Possible link between diabetes and cancer.

Deficiency of IQGAP2, a scaffolding protein expressed primarily in liver leads to rearrangements of hepatic protein compartmentalization and altered regulation of enzyme functions predisposing development of hepatocellular carcinoma and diabetes. Employing a systems approach with proteomics, metabolomics and fluxes characterizations, we examined the effects of IQGAP2 deficient proteomic changes on cellular metabolism and the overall metabolic phenotype. Iqgap2-/- mice demonstrated metabolic inflexibility, fasting hyperglycemia and obesity. Such phenotypic characteristics were associated with aberrant hepatic regulations of glycolysis/gluconeogenesis, glycogenolysis, lipid homeostasis and futile cycling corroborated with corresponding proteomic changes in cytosolic and mitochondrial compartments. IQGAP2 deficiency also led to truncated TCA-cycle, increased anaplerosis, increased supply of acetyl-CoA for de novo lipogenesis, and increased mitochondrial methyl-donor metabolism necessary for nucleotides synthesis. Our results suggest that changes in metabolic networks in IQGAP2 deficiency create a hepatic environment of a 'pre-diabetic' phenotype and a predisposition to non-alcoholic fatty liver disease (NAFLD) which has been linked to the development of hepatocellular carcinoma.

Identification of intrinsic order and disorder in the DNA repair protein XPA.

The DNA-repair protein XPA is required to recognize a wide variety of bulky lesions during nucleotide excision repair. Independent NMR solution structures of a human XPA fragment comprising approximately 40% of the full-length protein, the minimal DNA-binding domain, revealed that one-third of this molecule was disordered. To better characterize structural features of full-length XPA, we performed time-resolved trypsin proteolysis on active recombinant Xenopus XPA (xXPA). The resulting proteolytic fragments were analyzed by electrospray ionization interface coupled to a Fourier transform ion cyclotron resonance mass spectrometry and SDS-PAGE. The molecular weight of the full-length xXPA determined by mass spectrometry (30922.02 daltons) was consistent with that calculated from the sequence (30922.45 daltons). Moreover, the mass spectrometric data allowed the assignment of multiple xXPA fragments not resolvable by SDS-PAGE. The neural network program Predictor of Natural Disordered Regions (PONDR) applied to xXPA predicted extended disordered N- and C-terminal regions with an ordered internal core. This prediction agreed with our partial proteolysis results, thereby indicating that disorder in XPA shares sequence features with other well-characterized intrinsically unstructured proteins. Trypsin cleavages at 30 of the possible 48 sites were detected and no cleavage was observed in an internal region (Q85-I179) despite 14 possible cut sites. For the full-length xXPA, there was strong agreement among PONDR, partial proteolysis data, and the NMR structure for the corresponding XPA fragment.

Impact of body weight on urinary electrolytes in urinary stone formers.

OBJECTIVES: Obesity increases the risk of developing chronic medical conditions such as diabetes mellitus, hypertension, and coronary artery disease. We performed a retrospective review of a large data base on urinary stones to determine if differences are found in urine and serum chemistries among obese and nonobese stone-forming patients. The effect of body weight on stone recurrence among urinary stone formers was also determined. METHODS: A national data base containing serum biochemical profiles, 24-hour urine specimens, and standardized questionnaires was retrospectively evaluated from 5942 consecutive patients with urinary stone disease. Stone-forming patients were classified by body weight: nonobese men, less than 100 kg and nonobese women, less than 85 kg; intermediate men, 100 to 120 kg and intermediate women, 85 to 100 kg; and obese men, more than 120 kg and obese women, more than 100 kg. RESULTS: Obese stone formers comprised 6.8% (n = 404) of the patient population. The mean weight in the nonobese and obese groups was 81 kg versus 134 kg, respectively, for men and 64 kg versus 112 kg, respectively, for women. Obese patients represented 3.8% of the male and 12.6% of the female population. Obese patients had increased urinary excretion of sodium, calcium, magnesium, citrate, sulfate, phosphate, oxalate, uric acid, and cystine; obesity was associated with increased urinary volumes and urine osmolality compared with the nonobese patients. Obese men had increased concentration of urinary sodium, oxalate, uric acid, sulfate, and phosphate when corrected for urinary volume. Obese women had increased concentrations of sodium, uric acid, sulfate, phosphate, and cystine. The mean number of stone episodes in nonobese versus obese men was similar (3.55 and 3.56), whereas mean stone episodes were 2.93 and 3.38 (P = 0.045) for nonobese versus obese women. CONCLUSIONS: Among known stone formers, obesity is associated with unique changes in both serum and urinary chemistries. These changes are associated with an increased incidence of urinary stone episodes in obese women but not in obese men.

Obtaining more accurate Fourier transform ion cyclotron resonance mass measurements without internal standards using multiply charged ions.

Space-charge effects produce frequency shifts in Fourier transform ion cyclotron resonance (FTICR) mass spectrometry and correction for these shifts is necessary for obtaining accurate mass measurements. We report a novel method for obtaining accurate mass calibration to correct for space-charge induced mass shifts without the requirement for internal calibrants. The new approach is particularly well suited for electrospray ionization-FTICR mass spectra that contain multiple charge states of the same molecular species. This method, deconvolution of Coulombic affected linearity (DeCAL), is described and presented with several examples demonstrating the increased mass measurement accuracy obtained. DeCAL provides the basis for more routinely obtaining higher mass accuracy measurements in conjunction with chromatographic separations for complex mixture analysis, and obviates the need for internal calibration in many applications.

Accurate mass multiplexed tandem mass spectrometry for high-throughput polypeptide identification from mixtures.

We report a new tandem mass spectrometric approach for the improved identification of polypeptides from mixtures (e.g., using genomic databases). The approach involves the dissociation of several species simultaneously in a single experiment and provides both increased speed and sensitivity. The data analysis makes use of the known fragmentation pathways for polypeptides and highly accurate mass measurements for both the set of parent polypeptides and their fragments. The accurate mass information makes it possible to attribute most fragments to a specific parent species. We provide an initial demonstration of this multiplexed tandem MS approach using an FTICR mass spectrometer with a mixture of seven polypeptides dissociated using infrared irradiation from a CO2 laser. The peptides were added to, and then successfully identified from, the largest genomic database yet available (C. elegans), which is equivalent in complexity to that for a specific differentiated mammalian cell type. Additionally, since only a few enzymatic fragments are necessary to unambiguously identify a protein from an appropriate database, it is anticipated that the multiplexed MS/MS method will allow the more rapid identification of complex protein mixtures with on-line separation of their enzymatically produced polypeptides.

Protein identification with a single accurate mass of a cysteine-containing peptide and constrained database searching.

A method for rapid and unambiguous identification of proteins by sequence database searching using the accurate mass of a single peptide and specific sequence constraints is described. Peptide masses were measured using electrospray ionization-Fourier transform ion cyclotron resonance mass spectrometry to an accuracy of 1 ppm. The presence of a cysteine residue within a peptide sequence was used as a database searching constraint to reduce the number of potential database hits. Cysteine-containing peptides were detected within a mixture of peptides by incorporating chlorine into a general alkylating reagent specific for cysteine residues. Secondary search constraints included the specificity of the protease used for protein digestion and the molecular mass of the protein estimated by gel electrophoresis. The natural isotopic distribution of chlorine encoded the cysteine-containing peptide with a distinctive isotopic pattern that allowed automatic screening of mass spectra. The method is demonstrated for a peptide standard and unknown proteins from a yeast lysate using all 6118 possible yeast open reading frames as a database. As judged by calculation of codon bias, low-abundance proteins were identified from the yeast lysate using this new method but not by traditional methods such as tandem mass spectrometry via data-dependent acquisition or mass mapping.

A novel high-performance fourier transform ion cyclotron resonance cell for improved biopolymer characterization.

A new trapped ion cell design for use with Fourier transform ion cyclotron resonance mass spectrometry is described. The design employs 15 cylindrical ring electrodes to generate trapping potential wells and 32 separately assignable rod electrodes for excitation and detection. The rod electrodes are positioned internal to the ring electrodes and provide excitation fields that are thereby linearized along the magnetic field over the entire trapped ion volume. The new design also affords flexibility in the shaping of the trapping field using the 15 ring electrodes. Many different trapping well shapes can be generated by applying different voltages to the individual ring electrodes, ranging from quadratic to linearly ramped along the magnetic field axis, to a shape that is nearly flat over the entire trap volume, but rises very steeply near the ends of the trap. This feature should be useful for trapping larger ion populations and extension of the useful range of ion manipulation and dissociation experiments since the number of stages of ion manipulation or dissociation is limited in practice by the initial trapped ion population size. Predicted trapping well shapes for two different ring electrode configurations are presented, and these and several other possible configurations are discussed, as are the predicted excitation fields based on the use of rod electrodes internal to the trapping ring electrodes. Initial results are presented from an implementation of the design using a 3.5 T superconducting magnet. It was found that ions can be successfully trapped and detected with this cell design and that selected ion accumulation can be performed with the utilization of four rods for quadrupolar excitation. The initial results presented here illustrate the feasibility of this cell design and demonstrate differences in observed performance based upon different trapping well shapes.

Linkage of type II and type III cystinuria to 19q13.1: codominant inheritance of two cystinuric alleles at 19q13.1 produces an extreme stone-forming phenotype.

Cystinuria, a renal tubule disease affecting urinary cystine excretion with or without kidney stone formation, previously was mapped to chromosome region 2p.21. Mutations in the gene SLC3A1 or NBAT, the reported candidate gene for cystinuria at 2p.21, have been demonstrated in individuals with the autosomal recessive Type I cystinuria phenotype. Recently, the Type III cystinuria phenotype was mapped to chromosome region 19q13.1. Here we report a kindred of 39 persons in two families of cystinurics, Types II and III, that support linkage to 19q13.1 and exclude 2p.21. Based on a dominant model of inheritance, two-point analysis of the entire pedigree produced a maximum lod score (Z(max)) of 3.82 at marker D19S425. Multipoint analysis yielded a lod score of 4.96 at this marker, and a resultant lod score of 5.90 using a codominant model of inheritance. Furthermore, a candidate gene interval of 8.9 cM, flanked by markers D19S225 and D19S223, was obtained using multipoint and haplotype analyses. Thus, this kindred demonstrates the linkage of Type II cystinuria to 19q13.1 and confirms the linkage of Type III cystinuria at 19q13.1 while excluding the marker D19S225 that was previously included in the critical interval.

High-mass-measurement accuracy and 100% sequence coverage of enzymatically digested bovine serum albumin from an ESI-FTICR mass spectrum.

The application of Fourier transform ion cyclotron resonance (FTICR) mass spectrometry to the analysis of polypeptide mixtures resulting from proteolytic digestion is described. A new 11.5-T FTICR mass spectrometer has been applied for the analysis of tryptic digestion mixtures of the protein bovine serum albumin (BSA). The improved cyclotron frequency stability and reduced frequency shifts observed over a wide range of trapped ion population sizes provide the ability to signal average spectra without degrading mass measurement accuracy, requiring internal calibration or advanced data processing schemes to compensate for variations in ion cyclotron signals brought about by different population sizes. A total of 100 spectra were signal-averaged leading to the observation of a total of 123 isotope distributions with a signal-to-noise ratio greater than 3:1. From those distributions, 86 can be ascribed to tryptic fragments of BSA on the basis of mass measurement errors of 10 ppm or less. Of these, 71 were within 2 ppm error limits corresponding to complete amino acid sequence coverage and an average error of 0.77 ppm. These results indicate that high-accuracy measurements are feasible for a large number of species detected simultaneously without the necessity for internal calibration and indicate the potential of such measurements, when combined with chromatographic separations, for facilitating more rapid identification of large numbers of proteins.

Probing proteomes using capillary isoelectric focusing-electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry.

Unlike the genome, the proteome is exquisitely sensitive to cellular conditions and will consist of proteins having abundances dependent upon stage in the cell cycle, cell differentiation, response to environmental conditions (nutrients, temperature, stress etc.), or disease state(s). Therefore, the study of proteomes under well-defined conditions can provide a better understanding of complex biological processes and inference of protein function. Thus, much faster, more sensitive, and precise capabilities for the characterization of cellular constituents are desired. We describe progress in the development and initial application of the powerful combination of capillary isoelectric focusing (CIEF) and Fourier transform ion cyclotron resonance (FTICR) mass spectrometry for measurements of the proteome of the model system Escherichia coli. Isotope depletion of the growth media has been used to improve mass measurement accuracy, and the comparison of CIEF-FTICR results for the analysis of cell lysates harvested from E. coli cultured in normal and isotopically depleted media are presented. The initial studies have revealed 400-1000 putative proteins in the mass range 2-100 kDa from total injections of approximately 300 ng of E. coli proteins in a single CIEF-FTICR analysis.

The interaction between the chaperone SecB and its ligands: evidence for multiple subsites for binding.

The chaperone protein SecB is dedicated to the facilitation of export of proteins from the cytoplasm to the periplasm and outer membrane of Escherichia coli. It functions to bind and deliver precursors of exported proteins to the membrane-associated translocation apparatus before the precursors fold into their native stable structures. The binding to SecB is characterized by a high selectivity for ligands having nonnative structure but a low specificity for consensus in sequence among the ligands. A model previously presented (Randall LL, Hardy SJS, 1995, Trends Biochem Sci 20:65-69) to rationalize the ability of SecB to distinguish between the native and nonnative states of a polypeptide proposes that the SecB tetramer contains two types of subsites for ligand binding: one kind that would interact with extended flexible stretches of polypeptides and the other with hydrophobic regions. Here we have used titration calorimetry, analytical ultracentrifugation, and electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry to obtain evidence that such distinguishable subsites exist.

The observation of chaperone-ligand noncovalent complexes with electrospray ionization mass spectrometry.

Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) was applied for the study of noncovalent chaperone SecB-ligand complexes produced in solution and examined in the gas phase with the aid of electrospray ionization (ESI). Since chaperone proteins are believed to recognize and bind only with ligands with nonnative tertiary structure, this work required careful unfolding of the ligand and subsequent reaction with the intact chaperone (the noncovalent tetrameric protein, SecB). A high denaturant concentration was employed to produce nonnative structures of the OppA, and microdialysis of the resulting solutions containing the chaperone-ligand complexes was carried out to rapidly remove the denaturant prior to analysis. Multistage mass spectrometry was essential to the successful study of these complexes since the initial mass spectra indicated extensive adduction that precluded mass measurements, even after microdialysis. However, low energy collisional activation of the ions in the FTICR trap proved useful for adduct removal, and careful control of excitation level preserved the intact complexes of interest, revealing a 1:1 SecB:OppA stoichiometry. To our knowledge, these results present the first direct observation of chaperone-ligand noncovalent complexes and the highest molecular weight heterogeneous noncovalent complex observed to date by mass spectrometry. Furthermore, these results highlight the capabilities of FTICR for the study of such complex systems, and the development of a greater understanding of chaperone interactions in protein export.

In-trap cleanup of proteins from electrospray ionization using soft sustained off-resonance irradiation with fourier transform ion cyclotron resonance mass spectrometry.

Electrospray ionization Fourier transform ion cyclotron resonance (FTICR) mass spectrometry is capable of producing high mass resolving power and improved mass accuracy for large proteins and noncovalent complexes when coupled with collisionally induced dissociation (CID) of noncovalent adducts and consequent minimization of ion charge density in the ICR trap during measurements. This work demonstrates the application of in-trap cleanup to several biologically relevant systems, including carbonic anhydrase, 4-oxalocrotonate tautomerase (4OT) analogue, and SecB, a chaperone from Escherichia coli. In-trap cleanup yields improved mass measurements for these systems and is expected to further enable measurements for even more complex systems where adduction levels have precluded study of intact complexes.

Electrospray ionization-Fourier transform ion cyclotron resonance mass spectrometry at 11.5 tesla: instrument design and initial results.

Initial results obtained using a new electrospray ionization (ESI) Fourier transform ion cyclotron resonance (FTICR) mass spectrometer operated at a magnetic field 11.5 tesla are presented. The new instrument utilized an electrostatic ion guide between the ESI source and FTICR trap that provided up to 5% overall transmission efficiency for light ions and up to 30% efficiency for heavier biomolecules. The higher magnetic field in combination with an enlarged FTICR ion trap made it possible to substantially improve resolving power and operate in a more robust fashion for large biopolymers compared to lower field instruments. Mass resolution up to 10(6) has been achieved for intermediate size biopolymers such as bovine ubiquitin (8.6 kDa) and bovine cytochrome c (12.4 kDa) without the use of frequency drift correction methods. A mass resolution of 370,000 has been demonstrated for isotopically resolved molecular ions of bovine serum albumin (66.5 kDa). Comparative measurements were made with the same spectrometer using a lower field 3.5-tesla magnet allowing the performance gains to be more readily quantified. Further improvements in pumping capacity of the vacuum system and efficiency of ion transmission from the source are expected to lead to further substantial sensitivity gains.

Large Molecule Characterization Based upon Individual Ion Detection with Electrospray Ionization-FTICR Mass Spectrometry.

We report a new method for mass spectrometric measurements of high-molecular-weight species based on the summation of sequential Fourier transform ion cyclotron resonance (FTICR) spectra of individual multiply charged ions. This approach produces statistically useful mass spectra for large multiply charged molecular species formed by electrospray ionization and circumvents conventional limitations upon achievable resolving power and precision for high-molecular-weight species which arise due to Coulombic constraints. For very large molecules with tens to thousands of charges each, the total number of charges required to define the charge-state distribution, and thus provide accurate mass information, greatly exceeds the useful charge capacity of the FTICR cell. As trapped ion populations approach or exceed this capacity, FTICR performance degrades due to large frequency shifts, peak coalescence phenomena, and rapid loss of ion packet coherence, which effectively precludes high-resolution and precision measurements for molecules above ∼80-kDa size for a 7-T magnetic field strength. The present approach is based on the summation of many spectra having moderate populations of individual ions and relies on sensitivity sufficient for individual ion detection. While the number of trapped ions contributing to each mass spectrum may generally be insufficient to define the isotopic or charge-state distributions (and thus produce accurate information on the molecular weight distribution in a conventional fashion), the present data processing and summation approach suppresses the noise component (as well as smaller signals) that would otherwise be problematic. Importantly, this approach circumvents natural limitations for very high molecular weight species due to Coulombic interactions and thus provides a basis for much greater resolution and mass measurement accuracy than otherwise possible. This paper presents the details of this approach and its demonstration for the 66-kDa protein bovine serum albumin (where the conventional approach is also feasible) and discusses important aspects of the data manipulation.

Geriatric urolithiasis.

PURPOSE: We define the differences between geriatric patients with urinary stone disease compared to a younger cohort. MATERIALS AND METHODS: A data base, including serum biochemical profiles, 24-hour urinalyses and standardized questionnaires, was retrospectively evaluated from more than 6,000 consecutive patients with urinary stone disease. RESULTS: Geriatric stone formers comprised 12% (721) of all stone patients. Two-thirds of these elderly patients had aberrant urinary values and 29% had isolated hypocitraturia compared to 17% in the younger group. Of geriatric stone forming patients 76% had recurrent urinary stones (mean 3.5 stone episodes), which was similar to the younger comparable group (77%, mean 3.3 stone episodes). The severity of urinary stone disease was similar between the 2 groups based on the need for urological intervention. Geriatric stone patients, in general, experienced the first stone episode later in life (after age 50 years) compared with younger patients. Elderly patients had an increased incidence of uric acid stones, but had a similar incidence of struvite calculi. Geriatric stone patients underwent parathyroid surgery more frequently (2.7 versus 0.7%). Geriatric stone forming patients rarely had renal failure. CONCLUSIONS: The incidence, recurrence and severity of recurrent urinary stone disease were similar between geriatric and younger stone forming patients. Geriatric stone patients had an increased incidence of isolated hypocitraturia, uric acid calculi and previous parathyroidectomy. The geriatric stone population is not merely an extension of younger stone forming patients presenting at an older age. Rather, geriatric patients commonly experience the first symptomatic stone episode later in life.

A dual-trap design and its applications in electrospray ionization FTICR mass spectrometry.

A new arrangement consisting of two separate Fourier transform ion cyclotron resonance (FTICR) ion traps was used to develop methods for the manipulation of the ions produced by an electrospray ionization source (ESI). A first, "accumulation" trap, is generally maintained at a higher pressure than the second, high-performance "analyzer" trap. The manipulations developed and demonstrated include the following: (1) mass-selective ion transfers between the traps; (2) mass-selective step-wise accumulation of low-abundance ions of different mass-to-charge ratios transferred from the first trap to the analyzer trap; (3) simultaneous detection of ions in the analyzer trap and ion accumulation in the source trap; (4) simultaneous ion detection in the accumulation trap and ion storage in the analyzer trap; (5) sequential multiple transfers of the ions into the analyzer trap from the same ion population stored in the accumulation trap; (6) collision-induced dissociation of ions stored in the accumulation trap followed by mass-selective transfer of the product ions into the analyzer trap; (7) sequential transfer of the ions of different mass-to-charge ratios into the analyzer trap from the same ion population stored in the accumulation trap followed by the collision-induced dissociation of transferred ions in the analyzer trap. These ion manipulations benefit multistage studies and are projected to be useful in many biochemical applications of ESI-FTICR, including structural determination of biopolymers and study of noncovalent complexes.

Efficacy of radiographic imaging in pediatric blunt renal trauma.

PURPOSE: We sought to determine whether radiographic imaging can effectively detect significant renal injuries in children with blunt trauma who do not have significant hematuria. MATERIALS AND METHODS: We reviewed the records of 180 children who presented to our hospital for suspected renal trauma between 1977 and 1995. Results of excretory urography or abdominal computerized tomography were correlated with urinalysis findings and clinical outcome. RESULTS: Of 147 patients with microscopic hematuria after blunt trauma 77 underwent imaging. Only 1 patient had a significant renal injury (grade 2 or greater) and 76 had normal findings or renal contusions only, including 11 with microscopic hematuria and shock. Of the 74 patients who did not undergo imaging a clinical diagnosis of renal contusion was made and followup was available for 57 (77%). All patients healed without adverse sequelae. Of 33 patients with gross hematuria significant renal injuries were found in 9, including 3 who required immediate surgical repair of a major renal laceration or vascular injury. Combining our results with those of other reported series revealed significant renal injuries in only 11 of 548 children (2%) with less than 50 red blood cells per high power field on presenting urinalysis after blunt abdominal trauma. These patients were likely to have multiple associated injuries. CONCLUSIONS: Significant renal injuries are unlikely in pediatric patients with blunt renal trauma but no gross or substantial microscopic hematuria. Shock does not appear to be a clinically useful indicator.

Mass spectrometric characterization of sequence-specific complexes of DNA and transcription factor PU.1 DNA binding domain.

Electrospray ionization mass spectrometry (ESI-MS) has been used to study the noncovalent interaction of the 13.5-kDa DNA binding domain of PU.1 (PU.1-DBD) with specific double-stranded DNA (dsDNA) target molecules. Mixtures of PU.1-DBD protein and wild-type target DNA sequence yielded ESI-MS spectra showing only protein-dsDNA complex ions of 1:1 stoichiometry and free dsDNA. When PU.1-DBD protein, wild type target DNA, and a mutant target DNA lacking the consensus sequence were mixed, only the 1:1 complex with the wild-type DNA was observed, consistent with gel electrophoresis mobility shift assay results, demonstrating the observation of sequence-specific protein-dsDNA complexes using ESI-MS.