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2 A Phospho-Peptide Spectrum Library for Improved Targeted Assays
Overview
Purpose: Our long-term goal is quantification of peptides phosphorylated at specific
sites. We explore ways of designing appropriate targeted methods.
Methods: We acquire MS/MS spectra on synthetic phospho-peptides and compile
them into a spectrum library.
Results: Side-by-side spectrum comparisons of peptide sequences modified at
different residues reveal diagnostic fragments which can inform targeted assay
development.
Introduction
The profound and diverse effects of protein phosphorylation have created a keen
interest in their characterization and quantification. However, they present unique
challenges. Fragmentation of phosphorylated peptides is less predictable than
unmodified peptides, making more difficult the prediction of reliable fragment ions to
monitor in targeted assays. The presence of multiple isoforms that are not
chromatographically resolved present an additional complication in that one must rely
on specific product ions to distinguish between different modified forms since all forms
will share the same intact mass. To find diagnostic transitions, we turned to empirical
observation of synthetic phospho-peptides.
Methods
Sample Preparation
Synthetic peptides were prepared for 25 unique sequences containing between 2 and
6 different phosphorylation sites for each (Figure 1). They were mixed into pools of 8
peptides such that no two modified forms of the same sequence were mixed together.
Mass Spectrometry
Collision-induced dissociation (CID) and higher-energy C-trap dissociation (HCD)
tandem mass spectrometry (MS/MS) spectra were acquired on a Thermo Scientific™
LTQ Orbitrap™ mass spectrometer. Several spectra for each peptide were acquired by
sampling across the whole elution profile.
Data Analysis
Spectra were collected in a spectrum library that creates an averaged spectrum from
all observations from one peptide at each charge state and activation type. A software
tool was developed to predict and locate b- and y-type ions for all peptide isoforms in
the library spectra.
FIGURE 1. Synthetic peptides and their modifications. Red letter indicates a
phosphorylation site. A consolidated spectrum is built in the library when there
are at least five spectra acquired for a peptide. Some peptides were observed at
multiple charge states.
Results
Retention Time Does Not Re
We compared the retention ti
majority of peptides eluted wit
which may not be enough sep
(Figure 2).
FIGURE 2. Histogram of rete
peptides on a 60-minute gra
phosphorylated at position
phosphorylated at position
Library Consolidated Spectr
The spectrum library combine
peaks that are common to the
using a single observation to d
even in high-intensity fragmen
The library also provides a me
between isobaric peptides. Ide
different location of a phospho
is the only one predicted to diff
STFHAGQLR. The y8
2+ ion is
all peptides, we considered se
isoforms.
Phospopeptide sequence HCD Phospopeptide sequence HCD Phospopeptide sequence HCD
+2 +3 +4 +3 +2 +3 +4 +3 +2 +3 +4 +3
AGGKPSQSPSQEAAGEAVLGAK  RLSSTSLASGHSVR  SSSFREMDGQPER 
AGGKPSQSPSQEAAGEAVLGAK RLSSTSLASGHSVR S SSFREMDGQPER
AGGKPSQ SPSQEAAGEAVLGAK RLSSTSLASGHSVR S SSFREMDGQPER
AGGKPSQSP SQEAAGEAVLGAK SFNGSLKNVAVDELSR   SSSFREMDGQPER
ETTTSPKKYYLAEK  SFNGSLKNVAVDELSR SSSPTQYGLTK 
E TTTSPKKYYLAEK SFNGSLKNVAVDELSR S SSPTQYGLTK
ETTTSPKKYYLAEK SNSTSSMSSGLPEQDR  S SSPTQYGLTK
ETTT SPKKYYLAEK SNSTSSMSSGLPEQDR SSSPTQYGLTK
ETTTSPKKYYLAEK SNST SSMSSGLPEQDR SSSP T QYGLTK
FGESDTENQNNK  SNSTSSMSSGLPEQDR STFHAGQLR 
FGES DTENQNNK SNSTSS MSSGLPEQDR S TFHAGQLR
FGESDT ENQNNK SNSTSSMSSGLPEQDR S TFHAGQLR
FSDQAGPAIPTSNSYSK   SNSTSSMSSGLPEQDR STLVLHDLLK 
FSDQAGPAIPTSNSY SK SGGQRHSPLSQR  S TLVLHDLLK
FSDQAGPAIPTSNSYSK SGGQRHSPLSQR S TLVLHDLLK
GRRSPSPGNSPSGR  SGGQRHSPLSQR STVASMMHR 
GRRSPSPGNSPSGR SPGPSSPKEPLLFSR    S TVASMMHR
GRRSPS PGNSPSGR SPGPSSPKEPLLFSR STVASMMHR
ILSDVTHSAVFGVPASK   SPGPSSPKEPLLFSR VKEEGYELPYNPATDDYAVPPPR  
ILSDVTHSAVFGVPASK SQSDIFSR  VKEEGYELPY NPATDDYAVPPPR
ILSDVT HSAVFGVPASK SQSDIFSR VKEEGY ELPYNPATDDYAVPPPR
IQPSSPPPNHPNNHLFR   SQSDIFSR VQTTPPPAVQGQK 
IQPSSPPPNHPNNHLFR SRNSPLLER  VQ TTPPPAVQGQK
IQPSSPPPNHPNNHLFR SRNSPLLER VQTTPPPAVQGQK
LQTVHSIPLTINK   SRNSPLLER YIEDEDYYK 
LQTVHSIPLTINK SRTPPSAPSQSR   YIEDEDYYK
LQTVHSIPLTINK SRTPPSAPSQSR YIEDEDYY K
LRSADSENALSVQER   SRTPPSAPSQSR
LRSADSENALSVQER
LRSADS ENALSVQER
Consolidated spectrum Consolidated spectrum
CID CID
Consolidated spectrum
CID
Number of peptide isoform pairs