Biomedical subjects

D M Lubman

Publications and source records attributed to D M Lubman.

At least 19 recordsLinked to original sources

PubMed2003

Multiple sclerosis: an important role for post-translational modifications of myelin basic protein in pathogenesis.

Myelin basic protein (MBP) represents a candidate autoantigen in multiple sclerosis (MS). We isolated MBP from normal and MS human white matter and purified six components (charge isomers) to compare the post-translational modifications on each. The sites and extent of methylation, deimination, and phosphorylation were documented for all tryptic peptides by mass spectrometry. We found that mono and dimethylated arginine 107 was increased in MS samples; deimination of arginine occurred at a number of sites and was elevated in MS; phosphorylation was observed in 10 peptides in normal samples but was greatly reduced or absent in most peptides from MS samples. Data obtained with MBP isolated from fresh brain obtained from a spontaneously demyelinating mouse model supported the view that the changes observed in human brain were probably related to pathogenesis of demyelination, i.e. we found decreased phosphorylation and decreased amounts of glycogen synthesis kinase in brain homogenates using specific antibodies. This study represents the first to define post-translational modifications in demyelinating disease and suggest an important role in pathogenesis.

Animals↗

PubMed2001

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.

Cell Line↗

PubMed2001

Differential screening and mass mapping of proteins from premalignant and cancer cell lines using nonporous reversed-phase HPLC coupled with mass spectrometric analysis.

Nonporous (NPS) RP-HPLC has been used to rapidly separate proteins from whole cell lysates of human breast cell lines. The nonporous separation involves the use of hard-sphere silica beads of 1.5-microm diameter coated with C18, which can be used to separate proteins ranging from 5 to 90 kDa. Using only 30-40 microg of total protein, the protein molecular weights are detectable on-line using an ESI-oaTOF MS. Of hundreds of proteins detected in this mass range, approxinately 75-80 are more highly expressed. The molecular weight profiles can be displayed as a mass map analogous to a virtual "1-D gel" and differentially expressed proteins can be compared by image analysis. The separated proteins can also be detected by UV absorption and differentially expressed proteins quantified. The eluting proteins can be collected in the liquid phase and the molecular weight and peptide maps determined by MALDI-TOF MS for identification. It is demonstrated that the expressed protein profiles change during neoplastic progression and that many oncoproteins are readily detected. It is also shown that the response of premalignant cancer cells to estradiol can be rapidly screened by this method, demonstrating significant changes in response to an external agent. Ultimately, the proteins can be studied by peptide mapping to search for posttranslational modifications of the oncoproteins accompanying progression.

Cell Transformation, Neoplastic↗

PubMed2001

Chromatofocusing nonporous reversed-phase high-performance liquid chromatography/electrospray ionization time-of-flight mass spectrometry of proteins from human breast cancer whole cell lysates: a novel two-dimensional liquid chromatography/mass spectrometry method.

A novel two-dimensional two-column liquid chromatography/mass spectrometry (LC/MS) technique is described in this work, where chromatofocusing (CF) has been coupled to nonporous reversed-phase (NPS-RP) HPLC to separate proteins from human breast epithelial whole cell lysates. The liquid fractions from NPS-RP-HPLC are readily amenable to direct on-line analysis using electrospray ionization orthogonal acceleration time-of-flight mass spectrometry (ESI-TOFMS). A key advantage of this technique is that proteins can be 'peeled off' in the liquid phase from the CF column according to their isoelectric points (pI) in the first chromatographic separation dimension. The NPS-RP-HPLC column further separates these pI-focused fractions based upon protein hydrophobicity as the second chromatographic dimension. The third dimension involves on-line molecular weight determination using ESI-TOFMS. As a result, this method has the potential to be fully automated. In addition, a 2-D protein map of pI versus molecular weight is generated, which is analogous to a 2-D gel image. Thus, this technique may provide a means to study differential expression of proteins from whole cell lysates.

Breast↗

PubMed2001

Isoelectric focusing nonporous silica reversed-phase high-performance liquid chromatography/electrospray ionization time-of-flight mass spectrometry: a three-dimensional liquid-phase protein separation method as applied to the human erythroleukemia cell-line.

A liquid-phase three-dimensional protein separation method has been developed that is used to separate the cytosolic fraction of a HEL cell lysate via isoelectric focusing (IEF), nonporous silica (NPS) reversed-phase high-performance liquid chromatography (RP-HPLC) and electrospray ionization time-of-flight mass spectrometry (ESI-TOFMS), respectively. Several hundred unique protein molecular weights were observed in a pI range from 4.8 to 8.5 and a mass range from 5 to 85 kDa. Proteins were positively identified by analysis of the pI (+/-0.5 pI units), an intact protein molecular weight (+/-150 ppm), and peptide mass mapping results. Using the molecular weight (MW) and peptide mapping results of identified proteins it was possible to characterize their posttranslational (PTMs) and/or sequence modifications. PTMs were detected on both forms of cytosolic actin, heat shock 90 beta, HINT and alpha-enolase. Sequence modifications or conflicts were observed for beta-and gamma-actin, ATP beta-synthase and heat shock 90 beta. IEF-NPS-RP-HPLC/ESI-TOFMS was used to determine experimental pI, MW and relative hydrophobicity values for each protein detected. This data was used to generate a 2-D pI-MS protein map, where proteins are displayed according to their pI and molecular weight. Protein molecular weight peaks are represented as bands in the 2-D pI-MS image where the gray scale of each band is proportional to the intensity of the protein molecular weight peak. In addition, a third hydrophobicity dimension (%B) was added as the % acetonitrile elution to generate a 3-D pI-MS-%B plot where each protein can be tagged according to three parameters.

Amino Acid Sequence↗

PubMed2001

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.

Amino Acid Sequence↗

PubMed2000

Use of non-porous reversed-phase high-performance liquid chromatography for protein profiling and isolation of proteins induced by temperature variations for Siberian permafrost bacteria with identification by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and capillary electrophoresis-electrospray ionization mass spectrometry.

Non-porous reversed-phase high-performance liquid chromatography (NP-RP-HPLC) has been used to separate and isolate proteins from whole cell lysates of ED 7-3, a bacterium from the buried Siberian permafrost sediment. The proteins collected from the liquid eluent of this separation were then analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and capillary electrophoresis-electrospray ionization mass spectrometry (CE-ESI-MS). In order to study the differences in expression of cold-shock proteins (CSPs) at different growth temperatures, cultures of the ED 7-3 strain were prepared at 4 degrees C and 25 degrees C. The goals of this work were twofold: firstly, to identify the presence of CSPs and other proteins that are highly expressed at 4 degrees C but not at 25 degrees C; and secondly, to isolate these proteins for MALDI-TOF-MS and CE-ESI-MS identification. In this initial work, distinct protein profiles were observed for these cultures as a function of temperature. Fraction collection from the eluent of NP-RP-HPLC of some of the highly expressed proteins was performed and the proteins were mass analyzed for molecular mass. Peptide maps of the proteins were generated by tryptic digestion and were analyzed by CE-ESI-MS and MALDI-TOF-MS for database identification of the expressed proteins.

Amino Acid Sequence↗

PubMed2000

Isoelectric focusing nonporous RP HPLC: a two-dimensional liquid-phase separation method for mapping of cellular proteins with identification using MALDI-TOF mass spectrometry.

A novel two-dimensional liquid-phase separation method was developed that is capable of resolving large numbers of cellular proteins. The proteins are separated by pI using isoelectric focusing in the first dimension and by hydrophobicity using nonporous reversed-phase HPLC in the second dimension (IEF-NP RP HPLC). Proteins were mapped using original software in order to create a protein pattern analogous to that of the 2-D PAGE image. RP HPLC peaks are represented by bands of different intensity in the 2-D image, according to the intensity of the peaks eluting from the HPLC. Each peak was collected as the eluent of the HPLC separation in the liquid phase. The proteins collected were identified using proteolytic enzymes, MALDI-TOF MS and MSFit database searching. Using IEF-NP RP HPLC, approximately 700 bands were resolved in a pI range from 3.2 to 9.5 and 38 different proteins with molecular weights ranging from 12,000 to 75,000 were identified. In comparison to a 2-D gel separation of the same human erythroleukemia cell line lysate, the IEF-NP RP HPLC produced improved resolution of low mass and basic proteins. In addition, the proteins remained in the liquid phase throughout the separation, thus making the entire procedure highly amenable to automation and high throughput. It is demonstrated that IEF-NP RP HPLC provides a viable alternative to the 2-D gel separation method for the screening of protein profiles.

Chromatography, High Pressure Liquid↗

PubMed2000

Elucidation of peptide metabolism by on-line immunoaffinity liquid chromatography mass spectrometry.

An immunoaffinity chromatography extraction capillary liquid chromatography separation has been coupled to electrospray ionization mass spectrometry for on-line characterization of drug metabolites of a therapeutic peptide in plasma. It is demonstrated that the selectivity, sensitivity and molecular weight data provided by immunoaffinity chromatography coupled to liquid chromatography/mass spectrometry provides a means of rapidly achieving qualitative determinations of small amounts of material in complicated biological matrices such as plasma. The ability to detect the peptide in rat plasma at a level of 10 ng/mL is demonstrated using this method. In addition, experiments to study the epitope of the peptide by enzymatic digestion and mass spectrometry are also discussed. The method is proposed as an alternative approach to studying the metabolism of therapeutic peptides.

Amino Acid Sequence↗

PubMed2000

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.

Animals↗

PubMed2000

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.

Amino Acid Sequence↗

PubMed1999

Rapid profiling of induced proteins in bacteria using MALDI-TOF mass spectrometric detection of nonporous RP HPLC-separated whole cell lysates.

A method for rapid profiling of water-soluble proteins from whole cell lysates has been developed using matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry (TOFMS) following separation by reversed-phase high-performance liquid chromatography (RP HPLC). Rapid separation of proteins from cell lysates was achieved using columns packed with C18 nonporous (NP) silica beads. Using this method, the whole cell lysate water-soluble proteins of E. coli were separated in under 15 min. A method using two columns in series at different temperatures was used in order to provide high loadability without loss of separation efficiency. The nonporous packing in the columns provided for high recovery. Eluting fractions were collected and analyzed by MALDI-TOFMS to determine the molecular weights and peptide maps of the proteins. These methods provided for the rapid screening and identification of proteins from E. coli where the response of E. coli to L-arabinose induction was studied. In this work, it is demonstrated that NP RP HPLC with MALDI-TOFMS detection may serve as a rapid means of detecting and identifying changes in bacterial protein expression due to external stimuli.

Bacterial Proteins↗

PubMed1999

On-line capillary electrophoresis/microelectrospray ionization-tandem mass spectrometry using an ion trap storage/time-of-flight mass spectrometer with SWIFT technology.

The development of a system capable of the speed required for on-line capillary electrophoresis-tandem mass spectrometry (CE-MS/MS) of tryptic digests is described. The ion trap storage/reflectron time-of-flight (IT/reTOF) mass spectrometer is used as a nonscanning detector for rapid CE separation, where the peptides are ionized on-line using electrospray ionization (ESI). The ESI produced ions are stored in the ion trap and dc pulse injected into the reTOF-MS at a rate sufficient to maintain the separation achieved by CE. Using methodology generated by software and hardware developed in our lab, we can produce SWIFT (Stored Waveform Inverse Fourier Transform) ion isolation and TICKLE activation/fragmentation voltage waveforms to generate MS/MS at a rate as high as 10 Hz so that the MS/MS spectra can be optimized on even a 1-2 s eluting peak. In CE separations performed on tryptic digests of dogfish myelin basic protein (MBP) where eluting peaks 4-8 s wide are observed, it is demonstrated that an acquisition rate of 4 Hz provides > 20 spectra/peak and is more than sufficient to provide optimized MS/MS spectra of each of the eluting peaks in the electropherogram. The detailed structural analysis of dogfish MBP including several posttranslational modifications using CE-MS and CE-MS/MS is demonstrated using this method with < 10 fmol of material consumed.

Amino Acid Sequence↗

PubMed1999

Use of a mixed-mode packing and voltage tuning for peptide mixture separation in pressurized capillary electrochromatography with an ion trap storage/reflectron time-of-flight mass spectrometer detector.

A mixed-mode (reversed-phase/anion-exchange) stationary phase has been used as the capillary column packing for investigation of the separation of peptide mixtures in pressurized capillary electrochromatography (pCEC). This stationary phase contains both octadecylsilanes and dialkylamines. The amine groups of the stationary phase determine the charge density on the surface of the packing and can produce a strong and constant electroosmotic flow (EOF) at low pH. A comparison was made in terms of the capability of separating tryptic digests between the mixed-mode phase and C18 reversed phase. In addition, the constant EOF enabled the tuning of the retention and the selectivity of the separation by adjusting the mobile phase pH from 2 to 5. Furthermore, the magnitude and the polarity of the electric voltage were demonstrated to greatly influence the elution profiles of the peptides in pCEC. An ion trap storage/reflectron time-of-flight mass spectrometer was used as an on-line detector in these experiments due to its ability to provide rapid and accurate mass detection of the sample components eluting from the separation column.

Chromatography, Liquid↗

PubMed1999

Rapid screening of protein profiles of human breast cancer cell lines using non-porous reversed-phase high performance liquid chromatography separation with matrix-assisted laser desorption/ionization time-of-flight mass spectral analysis.

Non-porous reversed-phase (NP-RP) HPLC has been used to rapidly generate protein profiles of whole cell lysates of human breast cancer cell lines. The non-porous packing material used was silica coated with C18, which provided rapid separation with high collection efficiency of proteins from cell lysates. This method was used to study the differences in protein profiles among normal cells and fully malignant cells that share a common genetic background. The highly expressed proteins in each cell type were separated and collected in the liquid state where they were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) to obtain the molecular weight of the proteins. The protein fractions were then subjected to tryptic digestion and analyzed by pulsed delay extraction (PDE)-MALDI-TOFMS to obtain the peptide maps. The expressed proteins were identified based upon the molecular weight and peptide map using database-searching procedures. It is shown that key cancer-related proteins can be detected and identified which may be potentially used as biomarkers for cancer detection.

Biomarkers, Tumor↗

PubMed1999

Identification of proteins from two-dimensional gel electrophoresis of human erythroleukemia cells using capillary high performance liquid chromatography/electrospray-ion trap-reflectron time-of-flight mass spectrometry with two-dimensional topographic map analysis of in-gel tryptic digest products.

Protein spots from two-dimensional (2-D) gel electrophoresis of a human erythroleukemia cell line have been identified by analysis of the in-gel tryptic digests using capillary high performance liquid chromatography (HPLC) separation with on-line detection using electrospray ionization mass spectrometry (ESI-MS). This is performed using an electrospray/ion trap storage/reflectron time-of-flight mass spectrometer system (ESI-IT-reTOFMS). A 2-D topographic mapping display developed to process the on-line data acquired with this TOF system has been used to obtain mass identification of each peptide, even though the capillary HPLC only provides limited separation capability of the tryptic peptide mixtures studied herein. Using this method, a substantial fraction of the protein sequence can be covered and identified using the tryptic map. It is demonstrated that by entering the cell species, the approximate MW and pI range as determined by 2-D gel electrophoresis, and the tryptic peptide map into the database a unique match for identification of the protein generally results. It is also demonstrated that a much improved coverage of the protein sequence is obtained by this method relative to matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS).

Amino Acid Sequence↗

PubMed1999

Capillary electrophoresis/tandem mass spectrometry for analysis of proteins from two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis.

Capillary electrophoresis/time-of-flight mass spectrometry(CE/TOFMS) has been used for analysis of in-gel digests of protein spots excised from two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (2-D SDS-PAGE). An off-line purification and preconcentration procedure with a Zip Tip is used before CE/TOFMS analysis which allows for detection of protein spots with <1 picomole of material from 2-D gels. The off-line procedure provides sufficient purification for analysis while maintaining the quality of the CE separation. Using this procedure, several proteins from Coomassie Blue and zinc negatively stained gels are identified by the peptide maps generated and database searching. CE/TOF tandem mass spectrometry is used for the confirmation of database searching results and structural analysis of peptides that do not match the expected peptide maps obtained from the database in order to identify structural modifications. Several modifications were pinpointed and identified by this method.

Adenocarcinoma↗

PubMed1998

Separation of tryptic digests using a modified buffer in pressurized capillary. Electrochromatography with an ion trap storage/reflectron time-of-flight mass spectrometer.

A rapid and efficient separation method using pressurized capillary electrochromatography (pCEC) has been developed to separate protein digests. The effects of mobile-phase ion conductivity, pH, and column size on separation speed and column efficiency were studied and optimized. The pCEC method was demonstrated to provide enhanced speed with more efficient and selective separation than HPLC by performing separations of a bovine beta-lactoglobulin A digest. A human hemoglobin digest was separated by pCEC under optimized conditions, and more than 20 peaks were separated in less than 20 min. Using an ion trap storage/reflectron time-of-flight mass spectrometer, coeluting components were clearly identified, and the resolving power of the pCEC method was further enhanced.

Animals↗