A manual sequencing method for identification of phosphorylated amino acids in phosphopeptides

doi:10.1016/0003-2697(91)90356-X

Analytical Biochemistry

Volume 197, Issue 1, 15 August 1991, Pages 65-68

https://doi.org/10.1016/0003-2697(91)90356-X Get rights and content

Abstract

We describe a simple and inexpensive method for determining the location of phosphoamino acids in an isolated phosphopeptide. Phosphopeptides are immobilized on arylamine membrane discs using water-soluble carbodiimide, and the immobilized peptides are subjected to manual Edman degradation. The use of a membrane disc resulted in excellent recovery (65 to 80%) of radiolabeled phosphate following Edman degradation. Furthermore, interference from carryover and peptide washout is reduced to a minimum. The methodology should therefore be able to generate reliable results when used to analyze phosphopeptides that contain multiple phosphorylation sites. In addition to its advantage in sensitivity, the manual sequencing method is easy to perform and does not entail any sophisticated instrumentation.

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Fractions containing radioactivity were concentrated to less than 50 μl in a Speed-Vac. Manual and Automated Sequencing—Radiolabeled phosphopeptides obtained after peptide mapping or HPLC were brought to 65% acetonitrile and 0.1% trifluoroacetic acid in 20 μl, covalently attached to Sequelon-AA membrane discs, and subjected to manual Edman degradation as described (39). The membrane and the dried trifluoroacetic acid extracts were monitored for 32P release by Cerenkov counting after each cycle.
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Prior to amino acid sequencing, each phosphopeptide was immobilized on a disk of arylamine membrane (Sequelon-AA; Millipore), as described by the supplier. The disk was routinely cut into two parts; approximately 3/4 of the disk was applied to an amino acid sequencer (Procise 492; Applied Biosystems), while the remaining 1/4 was subjected to manual Edman degradation as described by others [18]. The computer program BLAST was utilized to match the identified phosphopeptide with sequences retrieved from the protein databases.
The identification of substrates is a key aspect in the study of the biological function of protein kinases. The procedure here described is aimed at profiling substrate phosphorylation at the phosphopeptide level by sequentially involving (i) the assessment of the in vitro activity of individual protein kinases on a complex mix of immobilized proteins, (ii) the fractionation of the phosphopeptides being released upon proteolysis of substrates, and (iii) the final identification of the targeted sequences. In particular, the protein sample is spotted onto nitrocellulose membrane and then subjected to a solid-phase kinase assay in the presence of [³²P]ATP, prior to solid-phase proteolytic digestion and two-dimensional phosphopeptide mapping. Radiolabeled phosphopeptides are subsequently isolated and sequenced to identify the substrates being targeted by the examined protein kinase. Using the γ-isotype of p21-activated protein kinase (γ-PAK) and its known in vitro substrates, I verified that both the specificity of substrate phosphorylation and its efficiency are similar upon solid- and liquid-phase conditions. To demonstrate the feasibility of the overall experimental system, I then employed a fairly crude cell extract as a source of candidate substrates and successfully identified the sequence of a putative substrate of γ-PAK.
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A manual sequencing method for identification of phosphorylated amino acids in phosphopeptides

Abstract

J. Biol. Chem

J. Biol. Chem

Anal. Biochem

J. Biol. Chem

J. Biol. Chem

J. Biol. Chem

J. Biol. Chem