enrichment and analysis of peptide subsets using fluorous affinity tags and mass spectrometry
TRANSCRIPT
Enrichment and analysis of peptide subsets usingfluorous affinity tags and mass spectrometryScott M Brittain, Scott B Ficarro, Ansgar Brock & Eric C Peters
Although mass spectrometry has become a powerful tool
for the functional analysis of biological systems, complete
proteome characterization cannot yet be achieved. Instead, the
sheer complexity of living organisms demands fractionation
of cellular extracts to enable more targeted analyses. Here,
we introduce the concept of ‘fluorous proteomics,’ whereby
specific peptide subsets from samples of biological origin are
tagged with perfluorinated moieties and subsequently enriched
by solid-phase extraction over a fluorous-functionalized
stationary phase. This approach is extremely selective, yet
can readily be tailored to enrich different subsets of peptides.
Additionally, this methodology overcomes many of the
limitations of traditional bioaffinity-based enrichment
strategies, while enabling new affinity enrichment schemes
impossible to implement with bioaffinity reagents. The
potential of this methodology is demonstrated by the facile
enrichment of peptides bearing particular side-chain
functionalities or post-translational modifications from tryptic
digests of individual proteins as well as whole cell lysates.
Functional proteome characterization involves the challenging task ofidentifying species of interest from among many thousands of pro-teins, each potentially altered by hundreds of possible post-translational modifications (PTMs). Additionally, living organismsoften exhibit large dynamic ranges in protein expression levels, rang-ing from estimated values of 104 in yeast to 109–1012 in plasma. As aresult of this extreme complexity, proteomics studies often use variousfractionation methodologies to focus on only a subset of the overallprotein complement. For example, numerous fractionation schemesbased on the presence of a particular chemical moiety such as a nativeamino acid side-chain functionality1,2 or a biologically importantPTM have been described3–8. Often, these affinity methods are specificfor a particular functionality, such as immobilized metal affinitychromatography for the enrichment of phosphorylated peptides6 orvarious lectins for the enrichment of specific glycosylated species9.Although highly effective, these approaches require the discovery anddevelopment of an individual reagent for each specific functionality.
Alternatively, a more generalized approach based on a family ofdual-functional reagents that possess different chemically reactivemoieties coupled to a common affinity capture moiety has beendescribed1,10,11. Typically, the classic biochemical affinity pair
biotin-streptavidin has been used to isolate the labeled species. How-ever, these custom reagents are relatively expensive, often difficult tofully elute from the capture resin12, and can complicate tandem massspectrometry (MS/MS) spectral interpretation4. As such, an ideal en-richment method would readily be tunable with respect to its chemicalselectivity, effect highly orthogonal but readily reversible isolations, behighly inert under MS/MS sequencing conditions and be economicalenough to use with samples of varying levels of complexity.
Since the introduction of fluorous biphasic catalysis techniques13,the field of fluorous chemistry has expanded rapidly. The term‘fluorous’ was coined to represent highly fluorinated (or perfluori-nated) species in a way analogous to how ‘aqueous’ represents water-based systems. It has been demonstrated that appending a shortperfluoroalkyl moiety to an organic compound enables the readyseparation of these fluorous species from other mixture componentsby solid-phase extraction over fluorous-functionalized silica gel (or,fluorous solid-phase extraction (FSPE)) because of the selectivityarising from the nature of fluorine-fluorine interactions14,15. Thismethodology enables the facile integration of synthesis and separation,and has been employed for the recycling and reuse of catalysts16,removal of reaction intermediates17 and the copurification of parallelsynthesis products18. Recently, fluorous protecting groups havealso aided in the purification of synthetic peptides19 and oligo-saccharides20. However, to date, these methodologies have been usedexclusively as processing aides for the targeted synthesis and purifica-tion of specific molecules in organic solvents.
Here, we demonstrate the use of fluorous affinity tags for the highlyefficient enrichment and subsequent mass spectrometric characteriza-tion of various subsets of peptides from highly complex mixtures ofbiological origin. The versatility of this methodology is demonstratedby the isolation of different classes of peptides bearing various side-chain functionalities or specific PTMs. Additionally, the uniqueproperties of these fluorous tags are utilized to implement fractiona-tion schemes that would be impossible to implement using traditionalbioaffinity tags.
Any viable proteomics fractionation methodology must enable boththe efficient isolation of a desired subset from the remainder of thesample as well as the subsequent efficient recovery and analysis of thatsubset. Such a fluorous proteomics strategy is outlined in Figure 1a.Although this scheme depicts selective labeling occurring at thepeptide level, it should be noted that the labeling of intact proteins
Published online 13 March 2005; doi:10.1038/nbt1076
Genomics Institute of the Novartis Research Foundation, 10675 John Jay Hopkins Drive, San Diego, California 92121, USA. Correspondence should be addressed toE.C.P. ([email protected]).
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followed by enzymatic digestion can also be performed, as discussedlater in the text. The resulting peptide mixture is loaded onto a FSPEcolumn, and nonlabeled peptides are removed from the column byisocratic washing with a solvent of suitable organic eluent concentra-tion. Tagged peptides are then selectively eluted with a higher organicconcentration and subjected to further analysis. Notably, all thevarious solutions used in this process are typically mixtures ofmethanol and water, enabling direct MS analysis of isolated peptideswith the exact composition of the eluent solution depending on boththe nature of the tag and the fluorous stationary phase used.
To demonstrate FSPE enrichment of tagged peptides from a single-protein digest, two peptides bearing a C8F17 group were added to atryptic digest of a-casein (Fig. 1b). This mixture was loaded onto anFSPE column and washed with 60% methanol, resulting in the elutionand collection of the nonlabeled peptides (Fig. 1c). Tagged specieswere then eluted from the column in 100% methanol. FSPE afforded asignificant enrichment of the tagged peptides (Fig. 1d). Importantly,this enrichment was affected by a simple solid-phase extractionmethod utilizing commercially available inexpensive reagents. Assuch, this methodology could be used cost effectively with a techniquesuch as two-dimensional gel electrophoresis that can separate proteinmixtures into thousands of different samples. Based on these promis-ing results, the ability of FSPE to enrich tagged peptides from far morecomplex mixtures was investigated. Thus, the same fluorous peptideswere spiked into a tryptic digest of Jurkat T–cell total protein extract,and the resulting mixture was subjected to the same FSPE enrichmentscheme. The tagged peptides were again easily isolated despite the fargreater complexity of the mixture (Fig. 2).
To assess the relative sensitivity and percent recovery of this affinitymethodology, the peptide VTQHFAK was synthesized and its freeN-terminal amino group was selectively reacted with 2H,2H,3H,3H-perfluoroundecanoic acid while still attached to the resin usingstandard peptide coupling conditions. The resulting purifiedfluorous-labeled peptide served as a convenient quantifiable standardin repeat spiking studies, and at the same time enabled an initialassessment of whether the site of modification affects the efficiency of
the isolation process. Thus, 20 pmol of the standard peptide was spikedinto 100 mg of a tryptic digest of Jurkat T–cell total protein extract, andthis mixture was subjected to the same FSPE enrichment scheme.Matrix-assisted laser desorption ionization (MALDI) analysis of one-tenth of the FSPE eluent demonstrated a similar dramatic enrichmentof the spiked peptide, whereas the percent recovery of the entireprocess measured between 50% and 55% (see Supplementary Fig. 1online and Supplementary Methods online for details). Additionally,500 fmol of the standard peptide was independently spiked into thesame quantity of tryptic digest, and after subjecting this mixture toFSPE, the fluorous peptide was again seen to be the dominant speciesin the MALDI mass spectrum of one-fifth of the FSPE eluent. Thesepreliminary results suggest that the fluorous affinity methodology,regardless of the site of modification, is indeed highly efficient.
We next sought to evaluate the compatibility of such fluoroustagged peptides with MS/MS sequencing strategies. Doubly chargedions of the fully tryptic peptides MPcF17TEDYLSLILNR (where cF17
represents a cysteine residue after reaction with N-[(3-perfluorooctyl)-propyl] iodoacetamide and MPcTEDYLSLILNR (where c representsa cysteine residue after reaction with iodoacetamide) were subjectedto MS/MS on an quadrupole time-of-flight mass spectrometer.The two MS/MS spectra are nearly identical (Fig. 3a,b) whentaking into account the clearly recognizable mass shift offluorous-labeled fragment ions, with the exception that the spectrumof the fluorous-modified peptide (Fig. 3a) exhibits a characteristicimmonium ion of monoisotopic mass 593.06 Da (labeled C�
I ) thatis absent in the spectrum of the traditionally alkylated peptide.The presence of this unique marker ion can thus readily serve asconfirmation that any given species is indeed labeled. Importantly,no ions corresponding to the loss or decomposition of the per-fluoroalkyl group were observed. Thus, fluorous affinity tags have adistinct advantage compared to numerous biotin-based reagents,which often suffer fragmentation during MS/MS and thus complicatespectral interpretation4.
Having shown the exquisite selectivity obtainable using fluorousaffinity tags, we next sought to demonstrate that the labeling reactions
Protein
Proteolysis
MS
Wash Elute
Fluorous-taggedpeptides
Non-taggedpeptides
MS
F
Selectiveintroduction of
fluoroustag
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qLsF17SGVSEIR QLsF17SGVSEIR
QLsF17SGVSEIR
qLsF17SGVSEIR Flow-through
Initialmixture
a
b c d
Figure 1 Fluorous proteomics strategy for the isolation of specific classes of peptides. (a) Depiction of the overall method. A specific peptide class is
selectively labeled with a fluorous tag (‘F’) bearing the appropriate functional group (for example, a thiol group to effect Michael addition after b-elimination)
and isolated using fluorous solid-phase extraction, followed by MALDI-MS or ESI-MS analysis. (b) MALDI-MS spectrum of a-casein tryptic digest spiked with
fluorous domain–containing peptides. (c) Nontagged peptides are observed in the MALDI-MS spectrum of the FSPE flow-through fraction. (d) Fluorous-tagged
peptides are observed in the MALDI-MS spectrum of the FSPE elution fraction. q, pyroglutamic acid; sF17, dehydroalanine residue after reaction with
1H,1H,2H,2H-perfluorodecane-1-thiol.
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could be performed directly on proteomics samples. Specifically, weinvestigated the enrichment of phosphorylated peptides from wholecell lysates utilizing established beta-elimination/Michael additionchemistry7,21,22. It should be noted that this reaction methodologysuffers from several well-documented limitations, including the inabil-ity to distinguish whether an identified site was initially phosphory-lated, modified with b-N-acetylglucosamine or in some cases wasoriginally an unmodified serine residue23. As such, other isolationmethodologies may be more efficient for the global profiling of suchmodifications5,24. Nevertheless, this study serves as a useful bench-mark of our affinity approach as several similar studies using tradi-tional bioaffinity-based reagents have already been reported.
Yeast total protein extract (400–500 mg) was digested with trypsin,and the resulting peptides were treated with performic acid tooxidize cysteine residues to cysteic acid. The oxidized peptides weresubjected to b-elimination/Michael addition using 1H,1H,2H,2H-perfluorooctane-1-thiol as the Michael donor, and subsequently
oxidized with H2O2 to generate a specific MS/MS marker foridentification12. The labeled peptides were enriched by FSPE andanalyzed by liquid chromatography (LC)/MS/MS. A total of 21candidate phosphorylated peptides were identified (SupplementaryTable 1 online), seven of which had previously been observed fromyeast total protein extract using a carbodiimide-based isolationstrategy3, indicating the validity of our approach. It should be notedthat the C6F13-containing thiol was used in this case so that theresulting tagged peptides were directly compatible with reversed-phaseLC columns typically used in proteomics studies. We observed that theC6F13 tag was slightly less selective than its C8F17 analog, as a smallnumber of nonphosphorylated peptides were detected (data notshown). However, these unlabeled peptides were often easy to recog-nize as such because of the absence of the sulfenic acid neutral loss.
Similarly, Jurkat T–cell total protein extract (5 mg) was treatedas described above, except that the tryptic peptides were firstprefractionated on a C18 reversed-phase medium after performic
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cqLsF17SGVSEIR
QLsF17SGVSEIR
Figure 2 Isolation of fluorous-tagged peptides from a highly complex
peptide mixture. MALDI-MS spectra of: (a) Jurkat cell total protein
digest spiked with fluorous domain–containing peptides, (b) flow-
through fraction after extensive washing with 50% acetonitrile and
(c) FSPE elution fraction. q, pyroglutamic acid; sF17, dehydroalanine
residue after reaction with 1H,1H,2H,2H-perfluorodecane-1-thiol.
* *
*
*
*
*
* *
***
**
MPcF17TEDYLSLILNR
[M+2H]2+
C Iy2a2
b12b11b10
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y13
a
b
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*
Figure 3 MS/MS spectra corresponding to (a) MPcF17TEDYLSLILNR
(cF17 represents a cysteine residue after reaction with N-[(3-perfluorooctyl)-
propyl] iodoacetamide), and (b) MPcTEDYLSLILNR (c represents a cysteine
residue after reaction with iodoacetamide); C�I , immonium ion of the
fluorous-labeled cysteine residue; �, loss of water/ammonia. MALDI-MS
spectra of (c) BSA tryptic digest after alkylation with N-[(3-perfluorooctyl)-
propyl] iodoacetamide and (d) after FSPE elution. �, fluorous tagged
cysteine-containing peptide.
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acid oxidation (eluent steps of 5%, 15%, 25% and 40% acetonitrile inwater). Each fraction was then individually subjected to b-elimination/Michael addition and treated with H2O2. The resulting tagged peptideswere enriched by FSPE and analyzed by LC/MS/MS. Nearly sixtycandidate phosphopeptides were identified (as shown in Supplemen-tary Table 2 online), 12% of which have been reported in the literature.Importantly, these two studies produced candidate phosphorylationsequences equal to or greater in number than those reported by otherstudies using chemically labeled affinity approaches3,4 despite the fargreater operational simplicity of the fluorous methodology.
The versatility of this enrichment strategy is greatly enhanced by thefact that it can readily be combined with a wide variety of highlyselective chemical functionalities. For example, reagents combining athiol-specific iodoacetamide moiety with a biotin affinity tag havebeen widely employed to simplify the complexity of a mixture beforemass spectrometric analysis by effecting the selective isolation ofcysteine-containing peptides1. By analogy, fluorous analogs of thesereagents would be expected to effect a similar enrichment. To test thissupposition, a tryptic digest of bovine serum albumin was reduced,treated with N-[(3-perfluorooctyl)-propyl] iodoacetamide and sub-jected to the same FSPE methodology described previously. As shownin Figure 3c,d, the tagged cysteine-containing tryptic peptides(marked ‘*’) could clearly be isolated (Fig. 3d) from their nonlabeledcounterparts. In fact, many of these cysteine-containing peptides werenot detected in the MALDI-MS analysis of the unfractionated digest(Fig. 3c). Similarly, the relative quantification capability of somebiotin-based reagents would similarly be expected to be affected byfluorous analogs having 13C substitutions in the fluorous domain,appropriate stable isotopic substitutions in the short linker regionbetween the reactive moiety and the fluorous domain (required toattenuate the inductive effect of the fluorines on the reactive moiety),or some combination thereof.
Although the examples presented so far have involved chemicallabeling of peptides, fluorous affinity reagents can also be used to
label intact proteins. Chicken ovalbumin was reduced and reactedwith N-[(3-perfluorohexyl)-propyl] iodoacetamide. The labeled pro-tein was then subjected to all the operations typical of gel-basedproteomics (that is, SDS-PAGE, in gel tryptic digestion and extractionof all resulting peptides from the gel whether labeled or not). Theextracted digest was analyzed both before and after FSPE by LC/MS/MS, and the results were searched using MASCOT allowing forappropriate variable modification of the cysteine residues (Supple-mentary Fig. 2a,b online, respectively). Although the same threefluorous-modified peptides were identified in both analyses, numer-ous peptides identified in the sample before FSPE were no longerdetected in the sample after FSPE despite the highly hydrophobicnature of some of these peptides. More importantly, MASCOTreturned similar scores for the MS/MS spectra of iodoacetamide orN-[(3-perfluorohexyl)-propyl] iodoacetamide-modified tryptic pep-tides (Supplementary Fig. 2c online), and thus is able to unequi-vocally identify the protein from the entire NCBInr database usingonly the MS/MS spectra of fluorous-modified peptides (Supplemen-tary Fig. 2d online), demonstrating that these affinity labels arecompletely compatible with commercially available protein identifica-tion software. Additional MS/MS spectra of fluorous-labeled peptidesare shown in Supplementary Figures 3–5 online.
In addition to the production of an entire family of more easilyused analogs of currently available labeling reagents, fluorous proteo-mic methodologies can also enable selective enrichment strategies thatcannot be implemented using classical bioaffinity pairs. For example,numerous PTMs including disulfide bonds or ubiquitination lead tothe production of branch sites within protein structures. After enzy-matic digestion, these sites of modification can be particularly difficultto detect since often the only discriminating feature between abranched peptide and the far more numerous linear peptides is thepresence of two N-terminal amino groups in the branched peptide.Given that it has been demonstrated that small molecules can beseparated based primarily on the length of their fluorous tag25, we
LRGG
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Figure 4 Separation of peptides based on fluorine content. MALDI-MS
spectra of (a) tryptic digest of polyubiquitin, (b) polyubiquitin tryptic digest
after guanidination of e-amino groups with O-methylisourea, (c) FSPE
elution fraction after derivatization of mixture with N-succinimidyl-3-
perfluorobutyl propionate and (d) MS/MS spectrum of the doubly labeled
‘‘Gly-Gly’’28 branched peptide.
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decided to investigate whether doubly tagged (branched) peptidescould be separated from singly tagged (linear) peptides using afluorous labeling strategy. Accordingly, a tryptic digest of polyubi-quitin (Fig. 4a) was used as a model system. The effective implemen-tation of the strategy requires the initial selective blocking of thee-amino groups of lysine residues. However, this can readily beaccomplished by guanidination (Fig. 4b)26,27. The resulting mixturewas reacted with the N-hydroxysuccinimide ester of 1H,1H,2H,2H-perfluorohexanoic acid, and the peptides were loaded onto an FSPEcolumn. Isocratic washing with 50% acetonitrile resulted in theelution of singly labeled peptides, whereas the doubly labeledbranched peptides were selectively eluted with pure methanol andsubsequently analyzed (Fig. 4c). Figure 4d shows the MS/MSspectrum of the doubly labeled ‘‘Gly-Gly’’28 branched peptide. Theseresults suggest that this strategy could be a general approach forthe study of these types of modifications that does not require theartificial transfection of affinity-tagged proteins into cell lines28, andis more selective than systems that rely solely on inducing changesin peptide hydrophobicity29.
We have described a methodology for the effective enrichment andsubsequent characterization of peptide subsets from complex mixturesof biological origin using fluorous affinity tags. Specifically, perfluor-oalkyl groups are selectively coupled to species of interest, and theresulting tagged peptides are readily isolated using a simple FSPEprocedure, the conditions of which facilitate their subsequent directcharacterization by mass spectrometry. The methodology is extremelyselective and has been demonstrated for the enrichment of differentclasses of peptides bearing various native side-chain functionalities(thiol, amino) or specific PTMs (phosphorylation). Additionally, themethodology can readily be extended to the enrichment of otherfunctional moieties owing to the facile synthesis of perfluoroalkylchains bearing a wide array of reactive functional groups. Further-more, the selectivity of the affinity enrichment can readily be tailoredby adjusting the nature of the fluorinated moiety incorporated intothe labeling reagent and/or the composition of the solid-phaseextraction medium. Thus, this methodology avoids many of theproblems associated with the use of classical bioaffinity strategies,including inefficient recovery of labeled substrates, fragmentationduring MS/MS and the relatively high cost of such reagents.
This methodology also enables separation schemes not possiblewith conventional bioaffinity reagents. For example, we are unaware ofany reports of peptides fractionated based on the number of biotin-based reagents with which they are labeled. By comparison, the abilityto achieve enrichment based on differing fluorine content readilyenables the fractionation of peptides bearing different numbers of aparticular functionality or PTM. Alternatively, different PTMs (forexample, phosphorylation and N-linked glycosylation) or functionalmoieties could be assayed in the same analysis by using affinity tagsthat differ in both their chemical reactivity and their fluorine content.
Future developments will undoubtedly extend the utility of thismethodology. For example, in addition to its relative inertness undercollision-activated dissociation conditions, fluorine exhibits a massdefect that could potentially be employed to enable the recognitionof tagged species based on high-mass-accuracy measurements. Alter-natively, the incorporation of cleavable linkers into the labelingreagents would enable highly selective enrichments to be followedby peptide characterization using classical high-performance liquidchromatography conditions. Finally, unlike classical bioaffinityreagents, this methodology could be directly applied to the selectiveisolation of different functional classes of small molecules inmetabolomic studies.
METHODSProteolysis. Proteolysis with sequencing-grade modified trypsin (Promega)
was carried out overnight at 37 1C using a substrate/enzyme ratio of
50:1 (wt/wt) in 100 mM ammonium bicarbonate. Bovine a-casein, serum
albumin and chicken ovalbumin were purchased from Sigma-Aldrich, and
polyubiquitin was purchased from Affiniti Research Products.
Preparation of yeast and Jurkat whole cell protein fractions. Yeast cake
(Saccharomyces cerevisiae) was purchased from a bakery supply store and
subsequently pulverized under liquid N2 and stored at �80 1C. Jurkat (E6-1,
ATCC) T cells were grown and harvested as described previously30. Cellular
protein was isolated using Trizol reagent (Invitrogen), and subsequently
oxidized by incubation overnight at 4 1C in 50 ml oxidation solution (4.5 ml
concentrated formic acid + 0.5 ml 30% H2O2). Finally, the oxidized protein
fraction was dialyzed into 100 mM ammonium bicarbonate and digested with
trypsin as described. Jurkat tryptic peptides were preparatively fractionated on
a C18 reversed-phase cartridge (Haisil, Higgins Analytical) using step eluent
solutions of 5%, 15%, 25% and 40% acetonitrile in 0.1% acetic acid.
Alkaline b-elimination/fluorous Michael addition to O-phosphopeptides.
Tryptic peptide digests (5 ml) were combined with an equal volume of 3:1
dimethyl sulfoxide/ethanol (vol/vol), followed by the addition of 4.6 ml
saturated Ba(OH)2 and 1 ml 500 mM NaOH. Finally, 0.7 ml of
1H,1H,2H,2H-perfluorooctae-1-thiol (Fluorous Technologies) was added and
the solution allowed to react at 37 1C for 1 h. The reactions were stopped by
acidification of the solution with 5% trifluoroacetic acid (vol/vol), and
subsequently oxidized by adjusting the solution to a final concentration of
3% H2O2 (vol/vol) and reaction for 30 min at room temperature (20 1C).
Fluorous derivatization of cysteinyl peptides. Bovine serum albumin (BSA)
tryptic digest was allowed to react with 50 mM N-[(3-perfluorooctyl)-propyl]
iodoacetamide (Fluorous Technologies, prepared as a 500 mM stock solution in
THF) in 25% ethanol (vol/vol) in 100 mM ammonium bicarbonate for 2 h in
the dark. Excess alkylating reagent was removed by incubation with 2 mg of
N-2-mercaptoethylaminomethyl polystyrene beads (Novabiochem) at room
temperature for 2 h.
Fluorous derivatization and processing of cysteinyl-containing protein.
Ovalbumin (B40 mM) was reduced with 10 mM TCEP in 6 M guanidinium
hydrochloride, 20 mM Tris, pH 8.0 buffer for 10 minutes at room temperature,
and reacted with 20 mM N-(1H,1H,2H,2H-perfluorohexyl)iodoacetamide for
1 h in the dark by addition of an equal volume of a THF solution of the
fluorous iodoacetamide. Excess reagents were removed using a disposable gel
filtration spin column packed with Biogel P6 beads (Micro Biospin P6,
Bio-Rad). The fluorous-labeled protein fraction was combined with an equal
volume of gel loading buffer (100 mM Tris, pH 6.8, 50 % glycerol, 0.1%
bromophenol blue, 1% SDS). SDS-PAGE was performed using a 150 V
constant voltage after loading several micrograms of derivatized protein into
each well of a 12 well, 1 mm � 8 cm � 8 cm 10–20% Tris-Glycine
polyacrylamide gel (Invitrogen). Following electrophoresis, the gel slab was
stained with colloidal Coomassie blue (Invitrogen) for 4 h, followed by
destaining in water overnight. Excised protein gel bands were cut into small
cubes (2–3 mm) and subjected to several wash and dehydration cycles (ten min
incubation with 50 ml of 100 mM ammonium bicarbonate, removal of the
liquid, ten min incubation with 25 ml acetonitrile and removal of the liquid).
The dehydrated gel cubes were vacuum dried for 5 min, rehydrated in 20 ml of
50 mM ammonium bicarbonate solution containing 10 ng/ml trypsin and
incubated at 37 1C overnight. Tryptic peptides were recovered by repeated
extraction (3�) with 80% acetonitrile/0.2% TFA (vol/vol).
Guanidination and N-terminal fluorous derivatization. Peptide lysine
e-amino groups were first converted to their homoarginine analogs. Tryptic
peptide digests were absorbed onto mC18 ZipTip (Millipore), and these tips
were then repeatedly aspirated in 10 ml of concentrated guanidination solution
(2 ml 0.5 M O-methylisourea hydrogensulfate dissolved in 8 ml 0.25 M sodium
carbonate, pH 11.7). The solution was then pipetted above the sorbent bed, and
the solution-imbibed tips were incubated for 2 h at 37 1C. Following
guanidination, peptides were thoroughly washed with 0.1% trifluoroacetic
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acid, eluted from the tips using 80% acetonitrile in 0.1% trifluoroacetic acid
and dried under vacuum. N-terminal a-amino groups were subsequently
derivatized by addition of an equal volume of 0.25 M sodium bicarbonate
buffer, pH 8.5, and freshly prepared 250 mM N-succinimidyl-3-perfluorobutyl
propionate in THF (40 ml total). The reactions proceeded for 2 h at room
temperature, followed by the addition of 4 ml of a 50% hydroxylamine solution
to reverse unwanted reactions. This solution was allowed to stand for 10 min,
followed by addition of 5 ml of 5% trifluoroacetic acid.
Fluorous Solid Phase Extraction (FSPE). FSPE was performed in packed fused
silica capillaries (360/200 mm O.D/I.D 5–8 cm length) containing fluorous
reversed-phase silica gel (FRPSG) (Fluoroflash Media; perfluorooctane bonded
phase, 5 mm particles, Fluorous Technologies). After equilibration with 60%
methanol (vol/vol) in 10 mM ammonium formate, a labeled sample was loaded
onto a column. The column was washed with multiple bed volumes of either
wash solution A (60% methanol (vol/vol) in 10 mM ammonium formate) or
wash solution B (50% acetonitrile (vol/vol)) depending on the fluorous tag
used (wash solution A used with C6F13 tags, wash solution B with C8F17 or
2xC4F9 tags). Finally, fluorous tagged peptides were eluted using B50 column
volumes of methanol.
Mass spectrometry and data analysis. MALDI-TOF analysis was performed
on a Bruker Biflex III mass spectrometer operated in the positive ion mode.
Peptides were deposited onto MALDI targets using the dried droplet method
by mixing with 2,5-dihydroxybenzoic acid matrix solution (10 mg/ml in 50%
acetonitrile with 0.2% trifluoroacetic acid). LC-ESI MS/MS was performed
using a Monitor C18 (Column Engineering) packed capillary column (3 mm
particles, 100 A, 75 mm I.D., 8 cm length, B5 mm spray tip) interfaced to a
hybrid quadrupole time-of-flight (QqTof) mass spectrometer (Micromass
Q-Tof-2) operating in survey scan mode. Tandem MS data were processed
and interrogated with the Mascot database search algorithm (Matrix Science).
Search parameters included the variable oxidation of cysteine to cysteic acid
and methionine to the methionine sulfone, as well as the C6F13 sulfoxide side-
chain modifications on former phosphoserine and phosphothreonine residues.
The latter two modifications were identified on the basis of their unique
residual masses together with recognition of their neutral loss products,
dehydroalanine (phosphoserine) or b-methyldehydroalanine (phosphothreo-
nine) in the MS/MS patterns.
COMPETING INTERESTS STATEMENTThe authors declare that they have no competing financial interests.
Note: Supplementary information is available on the Nature Biotechnology website.
Received 14 December 2004; accepted 26 January 2005
Published online at http://www.nature.com/naturebiotechnology/
1. Gygi, S.P. et al. Quantitative analysis of complex protein mixtures using isotope-codedaffinity tags. Nat. Biotechnol. 17, 994–999 (1999).
2. Shen, M. et al. Isolation and isotope labeling of cysteine- and methionine-containingtryptic peptides: application to the study of cell surface proteolysis. Mol. CellProteomics 2, 315–324 (2003).
3. Zhou, H., Watts, J.D. & Aebersold, R. A systematic approach to the analysis of proteinphosphorylation. Nat. Biotechnol. 19, 375–378 (2001).
4. Oda, Y., Nagasu, T. & Chait, B.T. Enrichment analysis of phosphorylated proteins as atool for probing the phosphoproteome. Nat. Biotechnol. 19, 379–382 (2001).
5. Ficarro, S.B. et al. Phosphoproteome analysis by mass spectrometry and its applicationto Saccharomyces cerevisiae. Nat. Biotechnol. 20, 301–305 (2002).
6. Posewitz, M.C. & Tempst, P. Immobilized gallium(III) affinity chromatography ofphosphopeptides. Anal. Chem. 71, 2883–2892 (1999).
7. Wells, L. et al. Mapping sites of O-GlcNAc modification using affinity tags for serineand threonine post-translational modifications. Mol. Cell Proteomics 1, 791–804(2002).
8. Zhang, H., Li, X.J., Martin, D.B. & Aebersold, R. Identification and quantification ofN-linked glycoproteins using hydrazide chemistry, stable isotope labeling and massspectrometry. Nat. Biotechnol. 21, 660–666 (2003).
9. Nilsson, C.L. Lectins: proteins that interpret the sugar code. Anal. Chem. 75, 348A–353A (2003).
10. Trester-Zedlitz, M. et al. A modular cross-linking approach for exploring proteininteractions. J. Am. Chem. Soc. 125, 2416–2425 (2003).
11. Campbell, D.A. & Szardenings, A.K. Functional profiling of the proteome with affinitylabels. Curr. Opin. Chem. Biol. 7, 296–303 (2003).
12. Steen, H. & Mann, M. A new derivatization strategy for the analysis of phospho-peptides by precursor ion scanning in positive ion mode. J. Am. Soc. Mass Spec.13, 996–1003 (2002).
13. Rabai, J. & Horvath, T. Facile catalyst separation without water: Fluorous biphasehydroformylation of olefins. Science 266, 72–75 (1994).
14. Curran, D.P. & Luo, Z. Fluorous synthesis with fewer fluorines (light fluorous synthesis):separation of tagged from untagged products by solid-phase extraction with fluorousreverse-phase silica gel. J. Am. Chem. Soc. 121, 9069–9072 (1999).
15. Curran, D.a.L., Zhiyong Fluorous techniques for the synthesis and separation of organicmolecules. Green Chem. 3, G3–G7 (2001).
16. Nakamura, Y., Takeuchi, S., Ohgo, Y. & Curran, D.P. Asymmetric alkylation of aromaticaldehydes with diethylzinc catalyzed by a fluorous BINOL-Ti complex in an organic andfluorous biophase system. Tetrahedron Lett. 41, 57–60 (2000).
17. Kainz, S., Luo, Z., Curran, D.P. & Leitner, W. Synthesis of perfluoroalkyl-substituted arylbromides and their purification over fluorous reverse phase silica. Synthesis 10, 1425–1427 (1998).
18. Luo, Z., Zhang, Q., Oderaotoshi, Y. & Curran, D.P. Fluorous mixture synthesis: afluorous-tagging strategy for the synthesis and separation of mixtures of organiccompounds. Science 291, 1766–1769 (2001).
19. de Visser, P.C. et al. A novel, base-labile fluorous amine protecting group: synthesis anduse as a tag in the purification of synthetic peptides. Tetrahedron Lett. 44, 9013–9016(2003).
20. Miura, T., Goto, K., Hosaka, D. & Inazu, T. Oligosaccharide synthesis on a fluoroussupport. Angew. Chem. Int. Ed. 42, 2047–2051 (2003).
21. Meyer, H.E., Hoffmann-Posorske, E., Korte, H. & Heilmeyer, L.M. Jr. Sequence analysisof phosphoserine-containing peptides. Modification for picomolar sensitivity. FEBSLett. 204, 61–66 (1986).
22. Byford, M.F. Rapid and selective modification of phosphoserine residues catalysed byBa2+ ions for their detection during peptide microsequencing. Biochem. J. 280 (Pt 1),261–265 (1991).
23. McLachlin, D.T. & Chait, B.T. Improved beta-elimination-based affinity purifi-cation strategy for enrichment of phosphopeptides. Anal. Chem. 75, 6826–6836(2003).
24. Khidekel, N., Ficarro, S.B., Peters, E.C. & Hsieh-Wilson, L.C. Exploring the O-GlcNAcproteome: direct identification of O-GlcNAc-modified proteins from the brain.Proc. Natl. Acad. Sci. USA 101, 13132–13137 (2004).
25. Curran, D.P. Fluorous reverse phase silica gel. A new tool for preparative separationsin synthetic organic and organofluorine chemistry. Synlett (9), 1488–1496 (2001).
26. Hale, J.E., Butler, J.P., Knierman, M.D. & Becker, G.W. Increased sensitivity of trypticpeptide detection by MALDI-TOF mass spectrometry is achieved by conversion of lysineto homoarginine. Anal. Biochem. 287, 110–117 (2000).
27. Peters, E.C., Horn, D.M., Tully, D.C. & Brock, A. A novel multifunctional labelingreagent for enhanced protein characterization with mass spectrometry. Rapid Commun.Mass Spectrom. 15, 2387–2392 (2001).
28. Peng, J. et al. A proteomics approach to understanding protein ubiquitination.Nat. Biotechnol. 21, 921–926 (2003).
29. Chen, X., Chen, Y.H. & Anderson, V.E. Protein cross-links: universal isolation andcharacterization by isotopic derivatization and electrospray ionization mass spectro-metry. Anal. Biochem. 273, 192–203 (1999).
30. Salomon, A.R. et al. Profiling of tyrosine phosphorylation pathways in human cellsusing mass spectrometry. Proc. Natl. Acad. Sci. USA 100, 443–448 (2003).
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