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developer of the AIDS Help project

Mass Spectrometry Proteomics:
Elements of Data Acquisition, Analysis and Interpretation

August 26th and 27th, 2006

All aspects of mass spectrometry in proteomics for the foreseeable future continue to be in a cycle of innovation and implementation. Keeping up with new techniques and ideas is important to be able to join in and contribute with new MS practices. The course discusses the theory and key points of three key elements driving innovations and their applications in biosciences today. The aims are to provide attendees with an orientation point from which to further use or initiate developments. A better understanding of peptide and protein fragmentation, full-scale quantitative analysis, and a clear representation and unambiguous validation of MS data will be provided to attendees. This short course will focus on the following aspects of mass spectrometry in proteomics:

1. Data acquisition (3 hours)

What types of fragmentation methods are available? What information do they provide and what sacrifices are given in terms of sensitivity and robustness? Both hot and cold fragmentation methods will be discussed; i.e. low and high energy CID, IRMPD, ECD and ETD as well as information on the instrumentation needed to conduct each type of fragmentation. This will be followed by applications using some of these fragmentation methods; e.g. top-down, bottom-up, and examples of analysis of post-translational modifications.

2. Data analysis (3 hours)

When should one use stable isotope labelling methods such as ICAT, iTRAQ or SILAC or newer, non-labelling methods based on statistical based methods for quantitation? Both in vitro chemical labeling and in vivo biological incorporation of stable isotopes will be reviewed from a perspective of the labels as well as instruments needed to acquire statistical sound data. Also, we will ask the question when is it appropriate to forgo labeling methods and use statistical analysis of ion intensity or peptide count to obtain quantitative data? The theoretical aspects will be followed by examples where quantitative proteomics helped solve biological problems using some of the commonly available quantitative methods.

3. Data interpretation (3 hours)

Data validation comes in many forms: from the use of multiple search engines, to tools for protein pathway analysis. We will review some commonly available search engines, data validation and data integration tools. Other areas that will be addressed include: data conversion, data validation. Questions such as the following will be addressed:

  1. what search engines work best for the novice and/or expert?
  2. under what circumstances should more than one search be used?
  3. how successful can one do de novo sequence analysis using algorithms as opposed to manual interpretation?
  4. how much does high mass accuracy of fragment ions help data interpretation?

Attendees will gain a better understanding of which strategies and tools can be used now with success and minimum input from experts outside their own laboratories and which should (for now) be left to collaborations with specialists. Each of three major sessions will be divided into two sub-sections of 1.5 hours each where methods will first be described didactically and then followed by discussion of specific applications in biology and medicine from expert lecturers attending the IMSC, allowing ample time for discussion, immediately or in the following days.

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