Metabolism of an insect diuretic hormone by Malpighian tubules studied by liquid chromatography coupled with electrospray ionization mass spectrometry

The larger of two diuretic hormones of the tobacco hornworm, Manduca sexta, (Mas-DH) is a peptide of 41 residues. It is one of a family of seven currently known insect diuretic hormones that are similar to the corticotropin-releasing factor–urotensin–sauvagine family of peptides. We investigated the possible inactivation of Mas-DH by incubating it in vitro with larval Malpighian tubules (Mt), the target organ of the hormone. The medium was analyzed, and degradation products were identified, using on-line microbore reversed-phase liquid chromatography coupled to electrospray ionization mass spectrometry (RPLC-ESI-MS). This sensitive technique allows identification of metabolites of Mas-DH (present at an initial level of ≈1 μM). An accurate Mr value for a metabolite is usually sufficient for unambiguous identification. Mas-DH is cleaved by Mt proteases initially at L29–R30 and R30–A31 under our assay conditions; some Mas-DH is also oxidized, apparently at M2 and M11. The proteolysis can be inhibited by 5 mM EDTA, suggesting that divalent metals are needed for peptide cleavage. The oxidation of the hormone can be inhibited by catalase or 1 mM methionine, indicating that H2O2 or related reactive oxygen species are responsible for the oxidative degradation observed. RPLC-ESI-MS is shown here to be an elegant and efficient method for studying peptide hormone metabolism resulting from unknown proteases and pathways.

The earliest archaeological maize (Zea mays L.) from highland Mexico: New accelerator mass spectrometry dates and their implications

Accelerator mass spectrometry age determinations of maize cobs (Zea mays L.) from Guilá Naquitz Cave in Oaxaca, Mexico, produced dates of 5,400 carbon-14 years before the present (about 6,250 calendar years ago), making those cobs the oldest in the Americas. Macrofossils and phytoliths characteristic of wild and domesticated Zea fruits are absent from older strata from the site, although Zea pollen has previously been identified from those levels. These results, together with the modern geographical distribution of wild Zea mays, suggest that the cultural practices that led to Zea domestication probably occurred elsewhere in Mexico. Guilá Naquitz Cave has now yielded the earliest macrofossil evidence for the domestication of two major American crop plants, squash (Cucurbita pepo) and maize.

DNA-bound transcription factor complexes analysed by mass-spectrometry: binding of novel proteins to the human c-fos SRE and related sequences

Transcription factors control eukaryotic polymerase II function by influencing the recruitment of multiprotein complexes to promoters and their subsequent integrated function. The complexity of the functional ‘transcriptosome’ has necessitated biochemical fractionation and subsequent protein sequencing on a grand scale to identify individual components. As a consequence, much is now known of the basal transcription complex. In contrast, less is known about the complexes formed at distal promoter elements. The c-fos SRE, for example, is known to bind Serum Response Factor (SRF) and ternary complex factors such as Elk-1. Their interaction with other factors at the SRE is implied but, to date, none have been identified. Here we describe the use of mass-spectrometric sequencing to identify six proteins, SRF, Elk-1 and four novel proteins, captured on SRE duplexes linked to magnetic beads. This approach is generally applicable to the characterisation of nucleic acid-bound protein complexes and the post-translational modification of their components.

Measurement of 8-hydroxy-2′-deoxyguanosine in DNA by high-performance liquid chromatography-mass spectrometry: comparison with measurement by gas chromatography-mass spectrometry

Measurement of 8-hydroxy-2′-deoxyguanosine (8-OH-dGuo) in DNA by high-performance liquid chromatography/mass spectrometry (LC/MS) was studied. A methodology was developed for separation by LC of 8-OH-dGuo from intact and modified nucleosides in DNA hydrolyzed by a combination of four enzymes: DNase I, phosphodiesterases I and II and alkaline phosphatase. The atmospheric pressure ionization-electrospray process was used for mass spectral measurements. A stable isotope-labeled analog of 8-OH-dGuo was used as an internal standard for quantification by isotope-dilution MS (IDMS). Results showed that LC/IDMS with selected ion-monitoring (SIM) is well suited for identification and quantification of 8-OH-dGuo in DNA at background levels and in damaged DNA. The sensitivity level of LC/IDMS-SIM was found to be comparable to that reported previously using LC-tandem MS (LC/MS/MS). It was found that approximately five lesions per 106 DNA bases can be detected using amounts of DNA as low as 2 µg. The results also suggest that this lesion may be quantified in DNA at levels of one lesion per 106 DNA bases, or even lower, when more DNA is used. Up to 50 µg of DNA per injection were used without adversely affecting the measurements. Gas chromatography/isotope-dilution MS with selected-ion monitoring (GC/IDMS-SIM) was also used to measure this compound in DNA following its removal from DNA by acidic hydrolysis or by hydrolysis with Escherichia coli Fpg protein. The background levels obtained by LC/IDMS-SIM and GC/IDMS-SIM were almost identical. Calf thymus DNA and DNA isolated from cultured HeLa cells were used for this purpose. This indicates that these two techniques can provide similar results in terms of the measurement of 8-OH-dGuo in DNA. In addition, DNA in buffered aqueous solution was damaged by ionizing radiation at different radiation doses and analyzed by LC/IDMS-SIM and GC/IDMS-SIM. Again, similar results were obtained by the two techniques. The sensitivity of GC/MS-SIM for 7,8-dihydro-8-oxoguanine was also examined and found to be much greater than that of LC/MS-SIM and the reported sensitivity of LC/MS/MS for 8-OH-dGuo. Taken together, the results unequivocally show that LC/IDMS-SIM is well suited for sensitive and accurate measurement of 8-OH-dGuo in DNA and that both LC/IDMS-SIM and GC/IDMS-SIM can provide similar results.

Attomole level protein sequencing by Edman degradation coupled with accelerator mass spectrometry

Edman degradation remains the primary method for determining the sequence of proteins. In this study, accelerator mass spectrometry was used to determine the N-terminal sequence of glutathione S-transferase at the attomole level with zeptomole precision using a tracer of 14C. The transgenic transferase was labeled by growing transformed Escherichia coli on [14C]glucose and purified by microaffinity chromatography. An internal standard of peptides on a solid phase synthesized to release approximately equal amounts of all known amino acids with each cycle were found to increase yield of gas phase sequencing reactions and subsequent semimicrobore HPLC as did a lactoglobulin carrier. This method is applicable to the sequencing of proteins from cell culture and illustrates a path to more general methods for determining N-terminal sequences with high sensitivity.

Direct measurement of oligonucleotide binding stoichiometry of gene V protein by mass spectrometry.

The binding stoichiometry of gene V protein from bacteriophage f1 to several oligonucleotides was studied using electrospray ionization-mass spectrometry (ESI-MS). Using mild mass spectrometer interface conditions that preserve noncovalent associations in solution, gene V protein was observed as dimer ions from a 10 mM NH4OAc solution. Addition of oligonucleotides resulted in formation of protein-oligonucleotide complexes with stoichiometry of approximately four nucleotides (nt) per protein monomer. A 16-mer oligonucleotide gave predominantly a 4:1 (protein monomer: oligonucleotide) complex while oligonucleotides shorter than 15 nt showed stoichiometries of 2:1. Stoichiometries and relative binding constants for a mixture of oligonucleotides were readily measured using mass spectrometry. The binding stoichiometry of the protein with the 16-mer oligonucleotide was measured independently using size-exclusion chromatography and the results were consistent with the mass spectrometric data. These results demonstrate, for the first time, the observation and stoichiometric measurement of protein-oligonucleotide complexes using ESI-MS. The sensitivity and high resolution of ESI-MS should make it a useful too] in the study of protein-DNA interactions.

Sequence verification of human creatine kinase (43 kDa) isozymes by high-resolution tandem mass spectrometry.

Amino acid sequencing by recombinant DNA technology, although dramatically useful, is subject to base reading errors, is indirect, and is insensitive to posttranslational processing. Mass spectrometry techniques can provide molecular weight data from even relatively large proteins for such cDNA sequences and can serve as a check of an enzyme's purity and sequence integrity. Multiply-charged ions from electrospray ionization can be dissociated to yield structural information by tandem mass spectrometry, providing a second method for gaining additional confidence in primary sequence confirmation. Here, accurate (+/- 1 Da) molecular weight and molecular ion dissociation information for human muscle and brain creatine kinases has been obtained by electrospray ionization coupled with Fourier-transform mass spectrometry to help distinguish which of several published amino acid sequences for both enzymes are correct. The results herein are consistent with one published sequence for each isozyme, and the heterogeneity indicated by isoelectric focusing due to 1-Da deamidation changes. This approach appears generally useful for detailed sequence verification of recombinant proteins.

Hydrophobic cluster analysis predicts an amino-terminal domain of varicella-zoster virus open reading frame 10 required for transcriptional activation.

Varicella-zoster virus open reading frame 10 (ORF10) protein, the homolog of the herpes simplex virus protein VP16, can transactivate immediate-early promoters from both viruses. A protein sequence comparison procedure termed hydrophobic cluster analysis was used to identify a motif centered at Phe-28, near the amino terminus of ORF10, that strongly resembles the sequence of the activating domain surrounding Phe-442 of VP16. With a series of GAL4-ORF10 fusion proteins, we mapped the ORF10 transcriptional-activation domain to the amino-terminal region (aa 5-79). Extensive mutagenesis of Phe-28 in GAL4-ORF10 fusion proteins demonstrated the importance of an aromatic or bulky hydrophobic amino acid at this position, as shown previously for Phe-442 of VP16. Transactivation by the native ORF10 protein was abolished when Phe-28 was replaced by Ala. Similar amino-terminal domains were identified in the VP16 homologs of other alphaherpesviruses. Hydrophobic cluster analysis correctly predicted activation domains of ORF10 and VP16 that share critical characteristics of a distinctive subclass of acidic activation domains.