Sequencing methods instruments

New instrumentation for the analysis of the proteome has been developed including a MALDI hybrid quadrupole time of flight instrument which combines advantages of the mass finger printing and peptide sequencing methods for protein identification (Andersen and Mann 2000). 

The advantages of on-line automation are the achievement of time savings in relation to the chromatographic method development time. The software can make decisions at any time of the day or night and can immediately communicate this information to the instrument after the completion of the experiment. There is also a more subtle benefit to the link of optimization software to the chromatography data system. Method development wizards with drop-down menus/user-defined fields can simplify the process of configuring the instrument sequence/method prior to a method development session. 

Mass spectrometry has been applied to the structure elucidation of peptides and proteins for some time. Depending on the problem, it is used as both a primary and a complementary technique and although many difficulties still remain it is now an established means of determining amino acid sequences. As instrumental methods and new derivatives have developed, the different approaches employed, with particular combinations of chemical pretreatment and sample introduction to the MS, have been dictated by the size and type of the peptide and the information required. 

Table I shows the present specifications of the commercial DNA sequencing instrument, which represents a tremendous jump over the capabilities of the normal sequencing methods. In the next section, I will briefly describe the major areas under investigation for continued optimization and improvement of this sequencing technology.

Most of the experimental information concerning copolymer microstructure has been obtained by physical methods based on modern instrumental methods. Techniques such as ultraviolet (UV), visible, and infrared (IR) spectroscopy, NMR spectroscopy, and mass spectroscopy have all been used to good advantage in this type of research. Advances in instrumentation and computer interfacing combine to make these physical methods particularly suitable to answer the question we pose With what frequency do particular sequences of repeat units occur in a copolymer. 

A sequence of tasks which combine the use of people, machines, methods, tools, environment, instrumentation, and materials to convert given inputs into outputs of added value. 

Sequencing of DNA is carried out by the Sanger dideoxy method, and small DNA segments can be synthesized in the laboratory by automated instruments. Small amounts of DNA can be amplified by factors of 106 using the polymerase chain reaction (PCR). 

Sample solution instability or incomplete extraction/separation would show up if several aliquots from the same sample work-up were put in a series of vials that would be run in sequence that would cover at least the duration of the longest sequence that could be accommodated on the autosample/instrument configuration. For example, if an individual chromatogram is acquired for 5.5 minutes, postrun reequilibration and injection take another 2.75 minutes, and 10 repeat injections are performed for each sample vial in the autosampler, then at least 15 60/(5.5 -I- 2.75)/10 = 11 vials would have to be prepared for a 5 P.M. to 8 A.M. (=15 hour) overnight run. If there is any appreciable trend, then the method will have to be modified or the allowable standing time limited. 

An ideal calibration curve (Figure 2.7) is a straight line with a slope of about 45 degrees. It is prepared by making a sequence of measurements on reference materials which have been prepared with known analyte contents. The curve is fundamental to the accuracy of the method. It is thus vitally important that it represents the best fit for the calibration data. Many computer software packages, supplied routinely with various analytical instruments, provide this facility. It is, however, useful to review briefly the principles on which they are based. 

In recent years, a novel approach to protein identification emerged, called top-down sequencing. Here the entire nondigested protein is analyzed. Apart from accurate MW measurement, the protein ion is fragmented by the electron capture dissociation (ECD) method (see Chapter 3). This provides in-depth information on the sequence of protein. Such analysis can be performed only with FTICR instruments (see Section 2.2.6) that ensure high resolution and accuracy but, at the same time, they are exceptionally expensive. However, as very large ions are analyzed, even the high accuracy of FTICR is sometimes not sufficient, and it is recommended that such analyses are accompanied by more traditional bottom-up approaches. 

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