Multiplexed isobaric tagging protocols for quantitative mass spectrometry approaches to auditory research.
Modern biologists have at their disposal a large array of techniques used to assess the existence and relative or absolute quantity of any molecule of interest in a sample. However, implementing most of these procedures can be a daunting task for the first time, even in a lab with experienced researchers. Just choosing a protocol to follow can take weeks while all of the nuances are examined and it is determined whether a protocol will (a) give the desired results, (b) result in interpretable and unbiased data, and (c) be amenable to the sample of interest. We detail here a robust procedure for labeling proteins in a complex lysate for the ultimate differential quantification of protein abundance following experimental manipulations. Following a successful outcome of the labeling procedure, the sample is submitted for mass spectrometric analysis, resulting in peptide quantification and protein identification. While we will concentrate on cells in culture, we will point out procedures that can be used for labeling lysates generated from other tissues, along with any minor modifications required for such samples. We will also outline, but not fully document, other strategies used in our lab to label proteins prior to mass spectrometric analysis, and describe under which conditions each procedure may be desirable. What is not covered in this chapter is anything but the most brief introduction to mass spectrometry (instrumentation, theory, etc.), nor do we attempt to cover much in the way of software used for post hoc analysis. These two topics are dependant upon one's resources, and where applicable, one's collaborators. We strongly encourage the reader to seek out expert advice on topics not covered here.
Department of Neuroscience, Tufts University School of Medicine, Boston, MA, USA.
Douglas E
DE
Vetter
Johnvesly
J
Basappa
Sevin
S
Turcan
D000818:Animals; D002460:Cell Line; D003051:Cochlea / Q000166:cytology / Q000378:metabolism; D007758:Ear, Inner / Q000166:cytology / Q000378:metabolism; D006801:Humans; D007553:Isotope Labeling / Q000379:methods; D013058:Mass Spectrometry / Q000295:instrumentation / Q000379:methods*; D011506:Proteins / Q000032:analysis*; D040901:Proteomics / Q000295:instrumentation / Q000379:methods*
D011506:Proteins