Identification Tools

Several tools are available to identify proteins using PMF. They all compare peptide masses obtained from mass spectrometry experiments to the theoretical peptide masses obtained from a theoretical digestion of aU sequences in a protein sequence database. 

The programs generate a list of protein entries, ordered by a score that tries to reflect the fit between theoretical and experimental parameters. It is therefore evident that the order of suggested proteins in the result hst is of paramount importance for a facilitated interpretation of the identification, in particular when manual intervention should be minimised. All programs compute scores for each hit some of these scoring systems are very simple, while others use probabilistic methods to increase confidence in the matching protein. A list of PMF tools and their URLs is given in Table 11. 

Protein attribute (MSorMSMS) Mowse Probabilistic models OWL http //srs.hgmp.mcr.ac.uk/ Pappin et at., 26 

Other tools SeqMS Prcd abilistic models htlp //www.protein.osaka-u. ac.j p/otganic/SeqMS. hunt F-Cossioerai, 167 

In the outputs of the software s that ranked the protein correctly, the score values and the discrimination values gave good hints about the quality of the match, particularly when comparing the first hit against the second one. One must consider 

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Research chemists cannot do these searches independently. There are a number of tools designed to identify and evaluate hazards. Several of these "identification tools are described below. 

The What if..method, the checklist, and HAZOP are well-publicized hazard identification tools. CCPS (1992) presents guidance on the use of these tools. 

The ready availability of carotenoid oxidation products through chemical methods will facilitate their use as standard identification tools in complex media such as biological fluids, and enable in vitro investigation of their biological activity. Moreover, these studies can help reveal the mechanisms by which they can be chemically or biochemically cleaved in vivo. 

To assure consistency and speed in multidisciplinary structure analysis of low-MW compounds involving various techniques (IR, NMR, MS, etc.) most industrial laboratories use a Standard Operating Procedure (SOP). In such schemes IR analysis is frequently used as a cheap filter for a quick starting control and as a means for verification. As IR detects only structural units identification of an unknown compound on the basis of IR is difficult. Mass spectrometry is used as the prime identification tool and is especially important in the determination of the exact mass and gross formulae. While structural prognostication on the basis of MS is difficult for the non-expert, a posteriori interpretation is quite feasible. H NMR is both easy and cheap, however requires greater sample quantities than either... 

Although often used as a qualitative (identification) tool, MS may act as a quantitative inorganic mass detector. Quantification of organic analytes often takes place in combination with chromatography or in tandem MS mode. It should be realised that mass spectrometry is certainly not a panacea for all polymer/additive problems, although it is developing into a major tool for this purpose. 

The What-if, the checklists and Hazop are well publicized hazard identification tools. But as Bollinger et al. (1996) have pointed out the use of any of these techniques demands knowledge, experience and flexibility. No prescriptive set of questions or key words or list is sufficient to cover all processes, hazards and all impacted populations. Bollinger et al. find that refinement of the quantitative measurement techniques such as safety indices and convergence to a single set of accepted indices would be beneficial. 

Also indices such as the Dow Fire and Explosion Hazard Index and the Mond Index have been suggested to measure the degree of inherent SHE of a process. Rushton et al. (1994) pointed out that these indices can be used for the assessment of existing plants or at the detailed design stages. They require detailed plant specifications such as the plot plan, equipment sizes, material inventories and flows. Checklists, interaction matrices, Hazop and other hazard identification tools are also usable for the evaluation, because all hazards must be identified and their potential consequences must be understood. E.g. Hazop can be used in different stages of process design but in restricted mode. A complete Hazop-study requires final process plans with flow sheets and PIDs. 

For these reasons, researchers have recently focused on developing faster robotic systems and more sensitive analytical metabolite identification tools [5-9]. However, such techniques are usually resource demanding, consuming a considerable amount of compound, and cannot be used before compound synthesis. Also, because of the increasing number of potential candidates, experimental metabolite identification remains a huge challenge. 

The use of surface-enhanced resonance Raman spectroscopy (SERRS) as an identification tool in TLC and HPLC has been investigated in detail. The chemical structures and common names of anionic dyes employed as model compounds are depicted in Fig. 3.88. RP-HPLC separations were performed in an ODS column (100 X 3 mm i.d. particla size 5 pm). The flow rate was 0.7 ml/min and dyes were detected at 500 nm. A heated nitrogen flow (200°C, 3 bar) was employed for spraying the effluent and for evaporating the solvent. Silica and alumina TLC plates were applied as deposition substrates they were moved at a speed of 2 mm/min. Solvents A and B were ammonium acetate-acetic acid buffer (pH = 4.7) containing 25 mM tributylammonium nitrate (TBAN03) and methanol, respectively. The baseline separation of anionic dyes is illustrated in Fig. 3.89. It was established that the limits of identification of the deposited dyes were 10 - 20 ng corresponding to the injected concentrations of 5 - 10 /ig/ml. It was further stated that the combined HPLC-(TLC)-SERRS technique makes possible the safe identification of anionic dyes [150],... 

R.M. Seifar, M.A.F. Altelaar, RJ. Dijkstra, F. Ariese, U.A. Th. Brinkman and C. Gooijer, Surface-enhanced resonance Raman spectroscopy (SERRS) as an identification tool in column liquid chromatography. Anal. Chem., 72 (2000) 5718-5724. 

Mass spectrometry, and especially LC/MS, is a major technique in the analysis of explosives. It combines good sensitivity and selectivity, and in addition to MS/ MS, provides an excellent identification tool for the forensic analyst. 

Causal factor identification tools are relatively easy to learn and easy to apply to simple incidents. For more complex incidents with complicated timelines, one or more causal factors can be overlooked, ultimately leading to missed root causes. Another disadvantage is that an inexperienced investigator could potentially assume that suppositions are causal factors, when in reality the supposed event or condition did not occur. 

Material response is typically studied using either direct (constant) applied voltage (DC) or alternating applied voltage (AC). The AC response as a function of frequency is characteristic of a material. In the future, such electric spectra may be used as a product identification tool, much like IR spectroscopy. Factors such as current strength, duration of measurement, specimen shape, temperature, and applied pressure affect the electric responses of materials. The response may be delayed because of a number of factors including the interaction between polymer chains, the presence within the chain of specific molecular groupings, and effects related to interactions in the specific atoms themselves. A number of properties, such as relaxation time, power loss, dissipation factor, and power factor are measures of this lag. The movement of dipoles (related to the dipole polarization (P) within a polymer can be divided into two types an orientation polarization (P ) and a dislocation or induced polarization. 

Despite its inherent analytical difficulties, gas chromatography on capillary columns in combination with sensitive and specific mass spectrometry has been widely used for separation of these analytes. Typical examples of such applications are those interfacing gas chromatography with mass spectrometry via electron impact (470, 484, 480, 489), chemical ionization (481, 478, 483, 473), or both interfaces (474, 475, 487, 488). Apart from mass spectrometry, Fourier transform infrared spectrometry has also been suggested as an alternative very useful identification tool in the area of the -agonist analysis. Capillary gas chromatography with Fourier transform infrared spectrometry was successfully employed to monitor clenbuterol, mabuterol, and salbutamol residues in bovine liver and urine (471). 

Mass chromatography is a new form of gas chromatography that uses two gas density detectors operated in parallel and provides (a) mass of components within 1-2% relative without determination of response factors, (b) molecular weight of components within 0.25-1% in the mass range 2—400, and (c) a powerful identification tool by the combined use of retention time and molecular weight data. The theoretical basis of the technique and its scope as a molecular weight analyzer, a qualitative identification tool, and a quantitative analyzer in the polymer field are discussed. 

In addition to the molecular techniques, technical advances both in chromatographic techniques and in identification tools, particularly the diverse forms of mass spectrometry, has allowed successful challenges to the separation and characterization of compounds of increasing complexity, poor stability, and low abundance [Whiting, 2001]. Information generated utilizing these techniques has resulted in characterization of a plethora of complex secondary metabolites that, in conjunction with the characterization of the enzymatic steps, has permitted the complete or partial elucidation of the flavonoid and the phenolic pathways present in many plants (Figs. 1.35 and 1.36). 

Compound identification in GC/MS methods is based on the retention time and the mass spectra interpretation the quantitation is based on the abundance of a primary (characteristic) ion. For a compound to be positively identified, all of the ions in the spectrum must be detected at one and the same retention time, which corresponds to the retention time of this compound in the calibration standard. The retention times of the primary ion and one or two secondary ions are typically monitored for this purpose. A combination of retention time and mass spectra is a formidable compound identification tool. That is why compound identification mistakes are relatively rare in GC/MS analyses, and they usually occur due to analysts lack of experience. 

Physiological fluid analysis by electrophoretic techniques is a very potent identification tool when supported by genetic population data (Stuver, Shaler, Marone, and Plankenhorn). McWright et al. report a careful study of the importance of environmental factors in determining the reliability of the genetic typing of bloodstains, another common clue material. 

A very useful identification tool is the combination of GC and thin-layer chromatography (TLC). The first work on combined GC-TLC appears to have been by Janak [59]. The GC column effluent is split into two streams, one of which enters the detector and the other, led via a heated conduit, impinges on the chromatographic thin layer carried by a moving plate. The GC fractions sampled in this way are subsequently developed and the TLC spots detected in the usual manner. The result is a kind of two-dimensional thin-... 

GC-MS with or without SIM is the most reliable identification tool of BRs.739 Prior to GC-MS analysis, vicinal hydroxyls in BRs are derivatized to methaneboronates, while isolated hydroxyl groups are converted to trimethylsilyl ethers (Figure 31). Fragment ions derived from the side chains have been used as diagnostic ions for mass spectrometric detection of BRs (Figure 31). Mass spectrometric data on methaneboronate derivatives of BRs have been reviewed.754 For quantitation of the endogenous levels of BRs, deuterated BRs have been frequently used as internal standards by adding to plant extracts.739... 

It is important to remember that changes in column packings and chromatographic systems may alter the retention of closely eluting compounds, and these elution schemes should only be used in conjunction with other identification tools such as tandem MS Ifagmen-tation patterns and high-mass accuracy measurements. 

The color of a mineral sample cannot be used to definitively identify the mineral because of impurities that may be present, however, the color can narrow down the identity of a mineral to a few choices. The streak of a mineral is the color of its powdered form. Rubbing the mineral across an unglazed porcelain square, called a streak plate, can best show streak color. A mineral will have a characteristic streak color, although more than one mineral may have the same color. Therefore, streak is not a definitive identification tool, although it may be used to verify the identity of a mineral of suspected composition. 

The high resolving power of FT-ICR-MS can readily be exploited in bottom-up protein identification. A nice example is the identification of high-abundant proteins in a tryptic digest of human plasma without any prior separation. The 2745 peaks in the spectmm could be reduced to 1165 isotopic clusters and 669 unique masses, 82 of which matched tryptic fragments of albumin (93% sequence coverage) and 16 others transferrin (41%) [39]. The same group showed that a theoretically predicted retention time of a tryptic peptide can be applied as an additional protein identification tool, next to its accurate mass acquired in LC-FT-ICR-MS. [40-41]... 

In our work at Leiden University, apart from HPLC-photodiode array (PDA) detection, HPLC-electrospray mass spectrometry was used as characterization and identification tools. A semipurified taxine extract obtained with acid/base extraction of T, baccata needles was ana-... 

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