HPLC–ES MS. Glycopeptides can be identified by characteristic fragment ions includ-
ing hexoseþ(163 Da) andN-acetyl hexosamineþ(204 Da).
Phosphoserine- and phosphothreonine-containing peptides can also be identified
by a process known asneutral loss scanningwhere these peptides show loss of 98 Da
byb-elimination of H 3 PO 4 (Fig. 9.25).9.6 Analysing protein complexes
Mass spectrometry is frequently used to identify partner proteins that interact with a
particular protein of interest. Interacting proteins can be isolated by a number of
methods including immunoprecipitation of tagged proteins from cell transfection;
affinity chromatography and surface plasmon resonance. Surface plasmon resonance
(SPR) (Section 13.3) technology has widespread application for biomolecular inter-
action analysis and during characterisation of protein–ligand and protein–protein
interactions, direct analysis by MALDI–TOF MS of samples bound to the Biacore chips
is now possible (where interaction kinetic data is also obtained; see Sections 13.3 and
17.3.2). Direct analysis of protein complexes by mass spectrometry is also possible. As
well as accurate molecular weight of large biopolymers such as proteins of mass greater
than 400 kDa, intact virus particles ofMr 40 106 (40 MDa) have been analysed using
ESI–TOF. An icosahedral virus consisting of a single-stranded RNA surrounded by a
homogeneous protein shell with a total mass of 6.5 106 Da and a rod-shaped RNA virus
with a total mass of 40.5 106 Da were studied on a ESI–TOF hybrid mass spectrometer.9.6.1 Sample preparation and handling
Mass analysis by ES–MS and MALDI–TOF is affected, seriously in some cases, by the
presence of particular salts, buffers and detergents. Keratin contamination from flakes of
skin and hair can be a major problem particularly when handling gels and slices; therefore
gloves and laboratory coats must be worn. Work on a clean surface in a hood with air filter
if possible and use a dedicated box of clean polypropylene microcentrifuge tubes tested to
confirm that they do not leach out polymers, mould release agents, plasticisers, etc.
Sample clean-up to remove or reduce levels of buffer salts, EDTA, DMSO, non-ionic and
ionic detergents (e.g. SDS) etc. can be achieved by dilution, washing, drop dialysis and ion
exchange resins. If one is analysing samples by MALDI–TOF, on-plate washing can
remove buffers and salts. Sample clean-up can also be achieved by pipette tip chromatog-
raphy (Section 11.2.5). This consists of a miniature C 18 reverse-phase chromatography
column, packed in a 10 nm^3 pipette tip. The sample, inlow or zero organic solvent-
containing buffer, is loaded into the tip witha few up- and-down movements of the pipette
piston to ensure complete binding of the sample. Since most contaminants described
above will not bind, the sample is trappedon the reverse phase material and eluted with a
solvent containing high organic solvent (typically 50–75% acetonitrile). This is particu-
larly applicable for clean-up of samples after in-gel digestion of protein bands separated
on SDS-PAGE. Coomassie Brilliant Bluedye is also removed by this procedure. The
technique can be used to concentrate samples and fractionate a mixture. Purification390 Mass spectrometric techniques