Michaela Scigelova, Gary Woffendin; Thermo Finnigan,
Hemel Hempstead, UK.
Malcolm Ward, Helen Byers; Proteome Sciences, London, UK.
Diane Hanger; Institute of Psychiatry KCL, London, UK.
The data presented here can be acquired using
any Thermo Finnigan LCQ Series ion trap mass spectrometer.
Phosphorylation of proteins plays a pivotal
role in the regulation of metabolic processes. Neurofilament proteins are
important structural features of the neuronal cytoskeleton. Phosphorylation
of these proteins is considered a critical factor for their assembly. Abnormal
phosphorylation of neurofilaments is associated with some neurodegenerative
conditions such as motor neuron disease, Parkinsons disease, and dementia.(1)
Determination of endogenous phosphorylation sites is therefore important
for understanding the role of neurofilament proteins in neurodegenerative
Previously, the analysis of phosphorylation sites relied largely on conventional
Edman sequencing following lengthy chromatographic separations or on two-dimensional
phosphopeptide mapping of proteins radiolabelled with phosphate.(2)
More recently, a technique for the sequencing of proteins directly from
polyacrylamide gels using electrospray in combination with tandem mass spectrometry
has been developed. This allows the exclusive use of automated mass spectrometry
techniques for sequencing and characterization of post-translational modifications
from total digest mixtures. In this regime, proteins are uniquely identified
by database searching of peptide fragmentation spectra.(3)
MS/MS spectra of peptides provide a wealth of information enabling the assignment
of peptide sequences and identification of modified residues. We present
a simple strategy enabling an unambiguous identification of the phosphorylated
residue of a peptide analyzed by LC/MS/MS in a complex peptide mixture.
In this report, we use capillary LC/MS/MS and
protein database searching techniques to:
- Identify a phosphorylated peptide by its neutral loss profile
- Unambiguously assign the modified amino acid residue
Thermo Finnigan Surveyor pump
Magic flow splitter (Michrom)
Thermo Finnigan LCQ Deca XP mass spectrometer fitted with NanoSpray Ion
Source (positive ion mode)
Data Dependent MS/MS
Capillary temp: 150C
Needle voltage: + 1.8 kV
LC column: PicoFrit BioBasic 4.9 cm,
75 um ID (New Objectives)
Injection volume: 2 L
LC solvent program:
Figure 1. Phosphorylated peptides exhibit
a prominent loss of a phosphate group in their MS/MS spectra. A loss of
49 amu observed in the MS/MS spectrum of a doubly-charged phosphorylated
peptide (RT = 27.71 min).
Proteins from a human brain sample were analyzed
using SDS-polyacrylamide gel electrophoresis, and the neurofilament proteins
were digested in-gel. A mixture of peptides was then analyzed by capillary
chromatography LC/MS, utilizing a fully-automated coupling of the Surveyor
LC and LCQ Deca XP. The data acquisition process included a full MS scan
followed by a full MS/MS scan of the most intense ion selected from the
preceding MS spectrum.
Phosphorylated peptides exhibit the loss of a phosphate group during fragmentation
in an ion trap (Figure 1). A prominent ion corresponding to a dephosphorylated
peptide is usually observed in the MS/MS spectrum. The neutral loss profile
display is used to locate a specific neutral loss in the acquired data (Figure
2). In this example, the loss of 49 amu corresponds to the difference between
the precursor ion mass of a doublycharged phosphorylated peptide and its
dephos-phorylated variant, observed in its MS/MS spectrum. This conveniently
highlights any possible phosphopeptide candidates within a given data set
Figure 2. Setting up a neutral loss profile.
Figure 3. A neutral loss profile detecting
a neutral loss of 49 amu (assuming doubly charged species) identified possible
phosphorylated candidate peptides from Figure 1. (A) Base peak chromatogram;
(B) Neutral loss profile.
The peptide is then identified by a TurboSEQUEST
database search that also confirms the exact location of the phosphorylation
residue within the peptide sequence. The TurboSEQUEST search engine, within
the BioWorks 3.0 protein identification software suite, can accommodate
multiple amino acid modifications in a single search routine. In this case,
an alkylation was considered for all cysteine residues in the sequence (a
static modification), while oxidation of methionine and phosphorylation
of serine and threonine were treated as differential modifications (Figure
Figure 4. TurboSEQUEST software can accommodate
multiple amino acid modifications in a single search routine.
The search provided an unambiguous identification
of the peptide (Figure 5), and confirmed the position of a phosphorylated
residue. Note that the assigned peptide sequence GVVTNGLDLSPADEK contains
two possible sites eligible for this modification (highlighted in blue).
TurboSEQUEST analysis of MS/MS data reveals the serine residue as the site
of phosphorylation (Figure 6).
Figure 5. Neurofilament protein identified
in TurboSEQUEST search. The peptide (in red) is phosphorylated at its serine
residue (highlighted in green).
Figure 6. Fragments in the MS/MS spectrum
of a phosphorylated peptide assigned by TurboSEQUEST search. The highlighted
peak (*) represents a doubly-charged fragment corresponding to a neutral
loss of phosphate from the original peptide.
We present here a simple strategy for the analysis
of phosphorylated peptides, which can be achieved on any Thermo Finnigan
LCQ series mass spectrometer. The combination of data dependent analysis
and advanced TurboSequest database searching identifies candidate phosphorylated
peptides in the LC/MS/MS data set, confirms the protein identity and sequence
of the peptide, and assigns the exact position of the phosphorylated amino
1. Betts, J.C., Blackstock, W.P., Ward, M.A.,
and Anderton, B.H. (1997) J. Biol. Chem. 272, 12922-12927, and references
2. Xu, Z.-S., Liu, W.-S., and Willard, M.B. (1992) J. Biol. Chem. 267, 4467-4471.
3. McCormack, A.L., Schieltz, D.M., Goode, B., Yang, S., Barnes, G., Drubin,
D., and Yates, J.R. (1997) Anal. Chem. 69, 767-776.
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