Protein Protocols Protein Protocols

610 Bonenfant, Mini, and Jenö
indicate the presence of several phosphorylated residues. The site of phosphorylation
in a given phosphopeptide can be identified by subjecting it to fragmentation in the
collision cell of the mass spectrometer. From the mass differences between the fragment
ions, the sequence can be “read” and the site of phosphorylation can be identified
when the mass difference is either 167, 181, or 243 Da, corresponding to the residue
masses of P-Ser, P-Thr, or P-Tyr. Because the activity of many proteins is regulated by
the extent to which they become phosphorylated by a particular stimulus, it is important
to be able to follow the extent of phosphorylation of a given site. Stoichiometries
can be quantified by comparison of the peak intensities of a given phosphopeptide and
its unphosphorylated counterpart.
Owing to its speed and sensitivity, it is evident that mass spectrometric tracing of
phosphorylation sites is a viable alternative to radioactive phosphopeptide mapping.
The main problem, however, lies in the complexity of the data. Proteins with large
molecular weights generate some 50 peptides on enzymatic digestion. In addition,
electrospray ionization produces multiple signals for each peptide due to multiple
charging. Therefore, data interpretation of phosphoprotein digests can be quite cumbersome.
Furthermore, phosphopeptides usually make up only a small portion of all
peptides in a digest. To reduce data complexity, procedures have been developed to
selectively detect phosphopeptides either by appropriate scanning procedures (13), or
by exploiting the phosphate group for selective phosphopeptide purification prior to
mass spectrometric analysis (14,15). This enables the researcher to find
phosphopeptides in crude protein digests with high speed and sensitivity.
In this chapter, we describe the basic steps of mass spectrometric phosphopeptide
mapping, namely enzymatic digestion of phosphoproteins, reverse-phase high-performance
liquid chromatography of peptides with mass spectral analysis (LC/MS), and
determination of the site of phosphorylation by tandem mass spectrometry (MS/MS).
2. Materials
2.1. Equipment
1. HPLC system: Two micro-gradient pumps are currently used in our laboratory: a Hewlett-
Packard 1090M (Palo Alto, CA), whose outlet is connected to a stainless steel T-piece to
reduce the flow to 1–2 µL/min and a splitless microgradient system consisting of an Evolution
200 system (Prolab, Reinach, Switzerland) operated at flow rates of 1 µL/min.
2. Model P-2000 quartz micropipet puller (Sutter Instrument Company, Novato, CA).
3. Capillary reverse-phase columns prepared from 100 µm inner diameter (i.d.) × 280 µm
outer diameter (o.d.) fused silica capillaries (LC Packings, Amsterdam, The Netherlands).
4. xyz-Positioner, model M-PRC-3 (Newport, Irvine, CA).
5. Stereo microscope, model Leica MZ12 (Wild Heerbrugg, Switzerland) and a CLS100
lamp (Wild Heerbrugg, Switzerland) equipped with two fiber-optic light guides installed
on the mass spectrometer.
6. Mass spectrometer: A TSQ7000 triple quadrupole instrument (Finnigan, San José, CA)
equipped with a homemade micro-electrospray ion source is used.
2.2. Materials and Reagents
1. Fused silica capillaries (100 µm i.d., 280 µm o.d.) and PEEK sleeves (300 µm i.d.) were
purchased from LC Packings (Amsterdam, The Netherlands). F120 PEEK Fingertight fittings
were from Upchurch Scientific (Oak Harbor, WA).