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Detection and Identification of Phosphorylation Sites in Proteins Using ,,, LC/MS/MS with Neutral Fragment Loss Mapping

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.

Protein Phosphorylation

The data presented here can be acquired using any Thermo Finnigan LCQ Series ion trap mass spectrometer. Introduction 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 disease.

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) The 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. Goal In this report, we use capillary LC/MS/MS and protein database searching techniques to:
  1. Identify a phosphorylated peptide by its neutral loss profile
  2. Unambiguously assign the modified amino acid residue
Experimental Conditions 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). Discussion 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 3). 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 4). 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. Conclusions 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 acid residue. References: 1. Betts, J.C., Blackstock, W.P., Ward, M.A., and Anderton, B.H. (1997) J. Biol. Chem. 272, 12922-12927, and references stated therein.
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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