Daniel B. Wall

Rapid profiling of E. coli proteins up to 500 kDa from whole cell lysates using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry was used to rapidly detect and profile large proteins from Escherichia coli whole cell lysates in the mass range 25-500 kDa. The bacterial samples were treated with guanidine hydrochloride and Triton X-100 to disrupt and solubilize the large inner membrane proteins. A sample preparation involving a nitrocellulose polymer film, and alpha-cyano-4-hydroxycinnamic acid, sinapinic acid or caffeic acid as matrix was utilized to rapidly monitor the presence of induced and repressed protein synthesis in response to L-arabinose catabolism in E. coli cells. The results were compared to those of 1-D or 2-D gel electrophoresis.

Mouse liver selenium-binding protein decreased in abundance by peroxisome proliferators.

Several studies with two‐dimensional gel electrophoresis (2‐DE) have shown that the abundance of numerous mouse liver proteins is altered in response to treatment with chemicals known to cause peroxisome proliferation. The peptide masses from tryptic digests of two liver proteins showing dramatic decreases in abundance in response to numerous peroxisome proliferators were used to search sequence databases. The selenium‐binding protein 2 (SBP2 formerly 56 kDa acetaminophen‐binding protein, AP 56) and selenium‐binding protein 1 (SBP1 formerly 56 kDa selenium‐binding protein, SP 56) in mouse liver, proteins with a high degree of sequence similarity, were the highest ranked identities obtained. Identity with SBP2 was subsequently confirmed by immunodetection with specific antiserum. Treatment of mice with 0.025% ciprofibrate resulted in the more basic of this pair of proteins being decreased to 30% of control abundance while the acidic protein was decreased to 7% of the control amount. Dexamethasone treatment, in contrast, caused increases of 80% and 20% in the abundance of the acidic and basic forms, respectively. Administration of dexamethasone to mice in combination with ciprofibrate produced expression of the acidic SBP2 at 23% of the control level and the basic SBP2 at 36%, a slightly moderated reduction compared with the decrease that occurred with ciprofibrate alone. These data suggest that peroxisome proliferators such as ciprofibrate cause a decrease in the abundance of the SBP2, which leads to increased cell proliferation, even in the presence of an inhibitor such as dexamethasone. Such a decrease in SBP, thought to serve as cell growth regulation factors, could be central to the nongenotoxic carcinogenicity of the peroxisome proliferators observed in rodents.

Rapid identification and screening of proteins from whole cell lysates of human erythroleukemia cells in the liquid phase, using non‐porous reversed phase high‐performance liquid chromatography separations of proteins followed by multi‐assisted laser desorption/ionization mass spectrometry analysis and sequence database searching

Non-porous reversed phase (NPRP) high-performance liquid chromatography (HPLC) has been used as a rapid method to separate proteins from whole cell lysates of human erythroleukemia (HEL) cells. Using phosphate-buffered saline (PBS) as a lysis buffer to extract proteins from HEL cells, more than 100 proteins of molecular weight up to 30 kDa were separated by the NPRP HPLC method, using a programmed acetonitrile:H2O gradient. The separated proteins were collected as liquid fractions as they eluted, and were further separated on the NPRP column with a different gradient to separate coeluting peaks. The isolated protein fractions were analyzed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) to determine the molecular weight of the protein. The proteins were cleaved by chemical or enzymatic digestion to produce peptide maps, which were analyzed by pulsed delayed extraction MALDI-MS. The peptide maps were matched against a database search to determine the protein identity. In some cases, several enzymes were used in order to find exactly one match against the database. This methodology is demonstrated for several proteins isolated from HEL cells and identified via database matching.

Studies of posttranslational modifications in spiny dogfish myelin basic protein

The objective of this investigation was to determine whether nonmammalian myelin basic protein contained charge isomers resulting from extensive posttranslational modifications as seen in mammalian MBP. Four charge isomer components from dogfish MBP have been isolated. These forms arise by phosphorylation and deamidation modifications. Components C1, C2 and C3 have been characterized. We are currently characterizing component C8. Dogfish MBP is less cationic than mammalian MBP and has about 50% lower mobility on a basic pH gel electrophoresis relative to human and to bovine MBP. The mammalian component C1, which is unmodified, is modified in the dogfish by phosphorylation. The reduced electrophoretic mobility is largely attributable to the charge reduction resulting from phosphorylation in serine 72, 83, and 120 or 121 in C1, and C3. In component C2, two or three phosphate groups were distributed among residues 134, 138 and 139. It was found that dogfish amino acid residue 30 was a lysine residue and not a glutamate residue as reported in the literature.

On-line capillary liquid chromatography tandem mass spectrometry on an ion trap/ reflectron time-of-flight mass spectrometer using the sequence tag database search approach for peptide sequencing and protein identification

Capillary high-performance liquid chromatography has been coupled on-line with an ion trap storage/reflectron time-of-flight mass spectrometer to perform tandem mass spectrometry for tryptic peptides. Selection and fragmentation of the precursor ions were performed in a three-dimensional ion trap, and the resulting fragment ions were pulsed out of the trap into a reflectron time-of-flight mass spectrometer for mass analysis. The stored waveform inverse Fourier transform waveform was applied to perform ion selection and an improved tickle voltage optimization scheme was used to generate collision-induced dissociation. Tandem mass spectra of various doubly charged tryptic peptides were investigated where a conspicuous y ion series over a certain mass range defined a partial amino acid sequence. The partial sequence was used to determine the identity of the peptide or even the protein by database search using the sequence tag approach. Several peptides from tryptic digests of horse heart myoglobin and bovine cytochrome c were selected for tandem mass spectrometry (MS/MS) where it was demonstrated that the proteins could be identified based on sequence tags derived from MS/MS spectra. This approach was also utilized to identify protein spots from a two-dimensional gel separation of a human esophageal adenocarcinoma cell line.

Comparison of the capabilities of liquid isoelectric focusing-one-dimensional nonporous silica reversed-phase liquid chromatography-electrospray ionization time-of-flight mass spectrometry and liquid isoelectric focusing-one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis mass mapping for the analysis of intact protein molecular masses

Nonporous silica reversed-phase HPLC coupled to electrospray ionization with on-line time-of-flight mass spectrometric detection (NPS-RP-HPLC-ESI-TOF-MS) is shown to be an effective liquid phase method for obtaining the molecular masses of proteins from pH fractionated cellular lysates where the method is capable of generating the same banding patterns typically observed using gel phase one-dimensional sodium dodecyl sulfatepolyacrylamide gel electrophoresis. The liquid-phase mass spectrometry-based method provides a mass accuracy of at least 150 ppm, with 4000 mass resolution and provides improved sensitivity as the protein molecular mass (MW) decreases. The liquid and gel phase methods are shown to be complementary in terms of their mass range but the liquid phase method has the advantage over the gel method in that the analysis times are 50 times shorter, the mass accuracy is 70 times better and the resolution is 130 times higher. The liquid phase method is shown to be more effective for detection of proteins below 40 kDa, while the gel phase separation can access many more proteins, including more hydrophobic proteins, at increasing MW.