Biological Mass Spectrometry

Biological mass spectrometry
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However, the Laboratory has access to several mass spectrometric technologies, including MALDI, thereby allowing investigators to adopt multiple analytical strategies. Biological Mass Spectrometry and Proteomics Mass spectrometry based proteomic experiments have become an important tool for studying biological systems. This site uses cookies. Accredited by the Higher Learning Commission. All trademarks are registered property of the University. Used by permission only.

Introduction to protein mass spectrometry

This page location is:. Sign In. The obvious solution is to start with enough sample ca.

Biological mass spectrometry: a primer.

But this requires high capacity fractionation and separation systems that generate a very large number of fractions from each sample ca. The conventional wisdom as expressed by commercial development of instrumentation and many of the leaders in biological mass spectrometry is that the future is electrospray, and MALDI is relatively unimportant.

We believe, on the contrary, that there are presently no known prospects for further dramatic improvement in electrospray MS technology while systems based on MALDI-TOF can provide powerful new tools for addressing the very difficult problems posed by large-scale applications of biological mass spectrometry.

Thus high sensitivity can be achieved but separation times are long and capacity severely limited.

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The main disadvantage of direct coupling between the separation and the mass spectrometer is that all of the measurements on an eluting peak must be made during the time that the peak is present in the effluent. Depending on the speed of the separation technique, this time may be as much as a minute or less than a second. Protein digests derived from complex biological extracts may contain many thousands of peptides in a single sample. Even after LC separation, hundreds of peptides may co-elute. Typically, measurements on these digests involve measurement of the peptide masses in MS mode, deciding which peaks should be measured using MS-MS, and measuring all of the MS-MS spectra of interest.

In many cases the separation must be slowed down to accommodate the speed of the mass spectrometer, or some of the potential information about the sample is lost. To interface MALDI with liquid separation techniques such as HPLC or CE, droplets from the liquid effluent, usually with added matrix solution, are deposited sequentially on a suitable surface and allowed to dry. Some systems have been described [ 14 ] for directly coupling MALDI with separations, but off-line coupling allows the sample deposition to occur at a speed appropriate to the chromatography, and the mass spectrometer can be operated faster or slower as needed to maximize the information.

For example, an entire LC run can be rapidly scanned in MS mode to determine the peptide masses and relative intensities for all peptides in the run. This information can then be used in a true data dependent manner to set up the MS-MS measurement for all of the spots on the plate to obtain the required information most efficiently. Since it rare for all of the sample to be used in most MALDI measurements, additional measurements can be made on the same plate at any later time as needed. Early applications of MALDI were limited by relatively low resolving power and mass accuracy of available TOF instruments, and the lack of a commonly available interface with liquid chromatography.

These developments are being pursued in ongoing research projects, and prototype instruments now provide speed, resolution, dynamic range, and sensitivity substantially superior to the performance available with commercially available instruments. In scanning instruments there is an additional efficiency term since only the measured peak is transmitted and all others are discarded. This term is unity for TOF, but not for conventional TOF-TOF since selection of a particular precursor requires rejection of all others in the peptide mass fingerprint spectrum.

The present generation of MALDI TOF instruments routinely operate at laser rates up to 5 kHz producing full mass spectra over any selected mass range following each laser shot.

It is very rare that a sufficient number of ions are produced in an individual laser shot to provide useful measurements of either peak centroids or intensities, and acquisition of such spectra over any significant time period quickly overwhelms the capabilities of even the most powerful computers.

Thus it is necessary to average a number of spectra using either a time-digital convertor TDC or transient digitizer with an on-board averaging. The principal advantage of higher laser rates is the flexibility that it provides to balance higher sample throughput against increased sensitivity, wider dynamic range, and better sample utilization. Producing spectra at these high rates may not be a problem, but storing and interpreting data at these rates is somewhat more demanding.

Under these conditions the typical data rate is ca. One day of continuous operation generates 8.

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Storing and managing this data stream is clearly not practical. On the other hand, all of the useful information in the spectrum is contained in the peak centroids, peak intensities, peak widths, and some measure of background noise. A typical averaged spectrum may contain on the order of useful peaks.

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If the important properties of each peak can be accurately expressed in 16 bytes 4 bit words , then the data rate is reduced by a factor of , and storage of only This is still substantial, but manageable with available hardware and software. We have recently developed novel algorithms that allow very rapid and accurate peak detection and mass calibration.

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The peak tables contain all the pertinent information about each peak including integrated intensity, centroid, and standard deviation. The new algorithms operate with the necessary speed without sacrificing accuracy as shown by preliminary results.

Biological Mass Spectrometry and Proteomics Facility

Biological Mass Spectrometry branding banner of the DNA‐binding cI repressor by electrospray ionization and fast atom bombardment mass spectrometry. Modern Mass Spectrometer (MS) Systems. Triple Quadrupole. MS systems used for proteomics have 4 tasks: • Create ions from analyte molecules. • Separate.

These peak tables constitute the raw data from the measurement and are written to disk. The raw time-of-flight data are discarded, but the peak tables contain sufficient data to allow a statistically equivalent raw TOF spectrum to be regenerated. These high data rates allow very rapid scanning and acquisition of tissue sections. In other applications speed may be less important than sensitivity, dynamic range, mass accuracy, and sample utilization.

One such application is LC-MS and MS-MS where the speed of the analysis is determined by the chromatograph and in some cases , laser shots may be used on some fractions. In these applications sensitivity and dynamic range are limited only by the chemical noise. This instrument also allows 10—50 fold multiplexing in MS-MS. This allows the generation of very high quality MS-MS spectra at unprecedented speed. All of the peptides present in a complex peptide mass fingerprint containing a hundred or more peaks can be fragmented and identified without exhausting the sample.

These specifications represent the current state of research instruments in our laboratory, and it should be noted that not all of these performance limits can be achieved simultaneously.

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Resolving power is independent of acquisition rate, but mass accuracy, dynamic range, and sensitivity depend on ion statistics that improve in proportion to the square root of the number of laser shots averaged. Our current sample plate accommodates fractions; thus either a series of very fast separations or parallel separations with longer retention time can be accommodated.

Recording high quality MS spectra from all fractions takes less than 10 minutes. The estimated analysis time may be modified as required by the results of the MS measurement, since some fractions may contain no interesting peaks and others may include a large number of relatively weak peaks requiring more laser shots. In our experience about , laser shots are required to desorb most of the sample in a fraction; thus the maximum time per fraction is 20 seconds.

Approximately 60, fractions per day can be analyzed with this approach. While these instruments and interfaces may provide the performance required for global analysis of biological samples, development of improved database and bioinformatics tools are essential for converting the massive volume of data that can be generated by these systems into useful information for addressing biological problems.

We believe that scientists can be sorted into three groups: users, innovators, and inventors, although some may span more than one group. We define users as those primarily interested in research on a particular problem or discipline doing the best work they can with available tools and protocols. Innovators discover new ways to use existing tools to address previously intractable problems, and inventors develop new tools.

The users constitute by far the largest group and are directly responsible for most of the measurable progress.

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The innovators generally receive the most attention and prestigious awards since they serve the vital function of adapting and demonstrating new tools for specific applications. The inventors are by far the smallest group, and they are largely ignored by users, innovators, manufacturers, and funding agencies until it is demonstrated by the innovators that the tools they invent are necessary for further advance of the science. The history of DNA sequencing leading to the genomic revolution offers some insight into the processes and interactions that lead to major advances, and this history may serve as a useful guide for similar revolutions in proteomics and metabolomics.

In , twenty-four years after the importance of DNA sequence was recognized, two general methods for DNA sequence analysis were developed independently by Sanger and Coulson[ 22 ] and Maxam and Gilbert [ 23 ]. These were adopted by many laboratories, and ten years later more than 15,, bases of DNA had been sequenced. Using these techniques a skilled biologist could produce about 50, bases of finished DNA sequence per year under ideal conditions.

In an automated approach to DNA sequencing was described [ 24 ], and the prospects for automated DNA sequencing and analysis of the human genome was reviewed by Hood, Hunkapiller, and Smith [ 25 ].