Unknown features in untargeted metabolomics and non-targeted analysis (NTA) are identified using ... more Unknown features in untargeted metabolomics and non-targeted analysis (NTA) are identified using fragment ions from MS/MS spectra to predict the structures of the unknown compounds. The precursor ion selected for fragmentation is commonly performed using data dependent acquisition (DDA) strategies or following statistical analysis using targeted MS/MS approaches. However, the selected precursor ions from DDA only cover a biased subset of the peaks or features found in full scan data. In addition, different statistical analysis can select different precursor ions for MS/MS analysis, which make the post-hoc validation of ions selected by new statistical methods impossible for precursor ions selected by the original statistical method. Here we propose an automated, exhaustive, statistical model-free workflow: paired mass distance-dependent analysis (PMDDA), for untargeted mass spectrometry identification of unknown compounds. By removing redundant peaks and performing pseudo-targeted M...
3551 Using SELDI-TOF mass spectrometric technique (SELDI=surface-enhanced laser desorption ioniza... more 3551 Using SELDI-TOF mass spectrometric technique (SELDI=surface-enhanced laser desorption ionization; TOF=time of flight) with H4-type chips, we have obtained protein profiles in plasma samples from patients with confirmed colon cancer (n=34) and control subjects (n=14). Peak mapping as well as gel viewing of the spectra revealed different expression levels at several masses. The most pronounced differences were observed for a peak at 8.9 kDa (8942.9±5.3), where the average differential expression of the intensities of the putative marker was approx. 3-fold higher in cancer patients (38.5±10.5) than in the controls (12.5±5.9). Molecular masses were determined to an accuracy of approx. 0.1%. In a second study (18 patients, 8 controls), the average differential expression of the intensities of the 8.9 kDa marker was approx. 2-fold higher in the cancer patients than in the controls. To identify this protein, plasma proteins were isolated with reversed-phase POROS R2 beads and further separated by SDS-PAGE. The protein bands of interest were excised from the gel and in-gel digested with trypsin. The resulting tryptic peptides were analyzed by μLC-electrospray-tandem (MS/MS), using an ion trap mass spectrometer. The MS/MS data were used to search a sequence and protein identification database (Knexus Sonar). Several candidates were found within the determined mass range, including complement component 3 precursor (C3). An analysis of the ion-chromatographic peak intensity of peptides corresponding to these candidates revealed that the highest peak intensities were associated with tryptic peptides of C3. The C3 is known to undergo multiple proteolytic hydrolysis and one of the hydrolysis products is the C3 peptide 672–748, known as anaphylatoxin C3a. The calculated molecular mass of C3a is 9094.6 Da. The average molecular mass of the 8.9 kDa peak is 8942.9 Da. Further examination of the tryptic peptides of C3 showed that all of the peptides identified were between the 713–736 of C3 sequence. All observed data suggested the 8.9 kDa protein to be a C3a-related protein. (Supported by T.J. Martell Foundation for Leukemia, Cancer and AIDS Research and NIH/NCI to R.W).
Unknown features in untargeted metabolomics and non-targeted analysis (NTA) are identified using ... more Unknown features in untargeted metabolomics and non-targeted analysis (NTA) are identified using fragment ions from MS/MS spectra to predict the structures of the unknown compounds. The precursor ion selected for fragmentation is commonly performed using data dependent acquisition (DDA) strategies or following statistical analysis using targeted MS/MS approaches. However, the selected precursor ions from DDA only cover a biased subset of the peaks or features found in full scan data. In addition, different statistical analysis can select different precursor ions for MS/MS analysis, which make the post-hoc validation of ions selected by new statistical methods impossible for precursor ions selected by the original statistical method. Here we propose an automated, exhaustive, statistical model-free workflow: paired mass distance-dependent analysis (PMDDA), for untargeted mass spectrometry identification of unknown compounds. By removing redundant peaks and performing pseudo-targeted M...
3551 Using SELDI-TOF mass spectrometric technique (SELDI=surface-enhanced laser desorption ioniza... more 3551 Using SELDI-TOF mass spectrometric technique (SELDI=surface-enhanced laser desorption ionization; TOF=time of flight) with H4-type chips, we have obtained protein profiles in plasma samples from patients with confirmed colon cancer (n=34) and control subjects (n=14). Peak mapping as well as gel viewing of the spectra revealed different expression levels at several masses. The most pronounced differences were observed for a peak at 8.9 kDa (8942.9±5.3), where the average differential expression of the intensities of the putative marker was approx. 3-fold higher in cancer patients (38.5±10.5) than in the controls (12.5±5.9). Molecular masses were determined to an accuracy of approx. 0.1%. In a second study (18 patients, 8 controls), the average differential expression of the intensities of the 8.9 kDa marker was approx. 2-fold higher in the cancer patients than in the controls. To identify this protein, plasma proteins were isolated with reversed-phase POROS R2 beads and further separated by SDS-PAGE. The protein bands of interest were excised from the gel and in-gel digested with trypsin. The resulting tryptic peptides were analyzed by μLC-electrospray-tandem (MS/MS), using an ion trap mass spectrometer. The MS/MS data were used to search a sequence and protein identification database (Knexus Sonar). Several candidates were found within the determined mass range, including complement component 3 precursor (C3). An analysis of the ion-chromatographic peak intensity of peptides corresponding to these candidates revealed that the highest peak intensities were associated with tryptic peptides of C3. The C3 is known to undergo multiple proteolytic hydrolysis and one of the hydrolysis products is the C3 peptide 672–748, known as anaphylatoxin C3a. The calculated molecular mass of C3a is 9094.6 Da. The average molecular mass of the 8.9 kDa peak is 8942.9 Da. Further examination of the tryptic peptides of C3 showed that all of the peptides identified were between the 713–736 of C3 sequence. All observed data suggested the 8.9 kDa protein to be a C3a-related protein. (Supported by T.J. Martell Foundation for Leukemia, Cancer and AIDS Research and NIH/NCI to R.W).
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Papers by Georgia Dolios