Liquid Chromatography-Fourier Transform Ion Cyclotron Resonance Mass Spectrometric Characterization of Protein Kinase C Phosphorylation Michael J. Chalmers,† John P. Quinn,† Greg T. Blakney,† Mark R. Emmett,†,‡ Harold Mischak,*,§ Simon J. Gaskell,| and Alan G. Marshall*,†,‡ National High Magnetic Field Laboratory, Florida State University, 1800 East Paul Dirac Drive, Tallahassee, Florida 32310-3706, Department of Chemistry and Biochemistry, Florida State University, Tallahassee, Florida 32310, Department of Nephrology, Medizinische Hochschule Hannover, Carl-Neuberg-Strasse 1, 30625 Hannover, Germany, and Michael Barber Centre for Mass Spectrometry, Department of Chemistry, UMIST, Manchester M60 1QD, United Kingdom Received January 31, 2003
A vented column, capillary liquid chromatography (LC) microelectrospray ionization (ESI) Fourier transform ion cyclotron resonance (FT-ICR (9.4 T)) mass spectrometry (MS) approach to phosphopeptide identification is described. A dual-ESI source capable of rapid (∼200 ms) switching between two independently controlled ESI emitters was constructed. The dual-ESI source, combined with external ion accumulation in a linear octopole ion trap, allowed for internal calibration of every mass spectrum during LC. LC ESI FT-ICR positive-ion MS of protein kinase C (PKC) revealed four previously unidentified phosphorylated peptides (one within PKCR, one within PKCδ, and two within PKCζ). Internal calibration improved the mass accuracy for LC MS spectra from an absolute mean (47 peptide ions) of 11.5 ppm to 1.5 ppm. Five additional (out of eight known) activating sites of PKC phosphorylation, not detected in positive-ion experiments, were observed by subsequent negative-ion direct infusion nanoelectrospray. Extension of the method to enable infrared multiphoton dissociation of all ions in the ICR cell prior to every other mass measurement revealed the diagnostic neutral loss of H3PO4 from phosphorylated peptide ions. The combination of accurate-mass MS and MS/MS offers a powerful new tool for identifying the presence and site(s) of phosphorylation in peptides, without the need for additional wet chemical derivatization. Keywords: Fourier transform • ion cyclotron resonance • ICR • FT-ICR • FTMS • phosphorylation • PKC • infrared multiphoton dissociation • tandem mass spectrometry • MS/MS • LC/MS • LC/MS/MS
Introduction Reversible phosphorylation is one of the most common protein post-translational modifications: approximately 30% of all proteins in eukaryotic cells are present in phosphorylated form at any given instant.1 The phosphorylation/dephosphorylation of proteins is performed by kinases and phosphatases respectively, and ∼2% of most eukaryotic genomes code for those enzymes.1,2 Phosphorylation is also one of the most important protein post-translational modifications and is responsible for the regulation of such critical biological processes as signal transduction. The sequencing of the human genome reveals that approximately 20% of human genes encode for proteins involved in signal transduction.2 Second Messenger-Activated Protein Kinase C (PKC). The second messenger-activated protein kinase C (PKC) family of * To whom correspondence should be addressed. † National High Magnetic Field Laboratory, Florida State University. ‡ Department of Chemistry and Biochemistry, Florida State University. § Department of Nephrology, Medizinische Hochschule Hannover. | Department of Chemistry, UMIST. 10.1021/pr030004d CCC: $25.00
2003 American Chemical Society
signal transducers is comprised of different isoforms that are divided into three subgroups according to their requirement for diacylglycerol (DAG) and calcium during activation. The classical PKCs (cPKCs: PKCR, PKCβ, and PKCγ) require both DAG and calcium for activation; the novel isoforms (nPKCs: PKCδ, PKC, PKCη, and PKCθ) require only DAG whereas the atypical PKCs (aPKCs: PKCζ, PKCι/λ) require neither DAG nor calcium for activation. PKCs have been implicated in various intracellular signaling processes. The different isozymes play major roles in such different biological processes as mitogenesis and transformation,3 differentiation,4 apoptosis,5,6 and migration.7 The regulation of all of the above PKCs through the binding of second messenger molecules has long been known.8 Recently, phosphorylation has been shown to be a critical mechanism for the structural, spatial and kinetic control of all three PKC classes. PKC “in statu nascendi” is essentially inactive. This inactive enzyme is initially phosphorylated by 3-phosphoinositide-dependent protein kinase-1 (PDK1) in the activation loop.9 Subsequently, PKC autophosphorylates at two C-terminal sites, resulting in a mature enzyme that is capable Journal of Proteome Research 2003, 2, 373-382
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Table 1. Three Main Sites of Phosphorylation Involved in the Activation of PKC8 a
isotype
classical R β1(II) β2(I) γ novel δ η θ atypical ζ ι
activation loop
C-terminal autophosphorylation
C-terminal hydrophobic site
Thr 497 TFCGT Thr 500 TFCGT Thr 500 TFCGT Thr 514 TFCGT
Thr 638 TPPDQ Thr 641 TPPDQ Thr 642 TPDPK Thr 655 TPPDR
Thr 657 FSYVN Ser 660 FSFVN Ser 661 FSYTN Thr 674 FTYVN
Thr 505 TFCGT Thr 566 TFCGT Thr 513 THCGT Thr 538 TFCGT
Ser 643 SFSDK Thr 710 TLVDE Thr 655 TPIDE Ser 676 SFADR
Ser 662 FSFVN Ser 729 FSYFG Ser 674 FSYVS Ser 695 FSFIN
Thr 410 TFCGT Thr 403 TFCGT
Thr 560 TPDDE Thr 574 TPDDD
(Glu 579) FEFIN (Glu 555) FEYIN
others
Thr 250 DWDRT
a Atypical PKCs contain a phosphomimetic glutamic acid residue in place of a serine or threonine residue at the C-terminal hydrophobic site. Other known sites of Ser/Thr phosphorylation are also listed.
of properly transmitting signals from second messengers.10 Three main sites of PKC phosphorylation have been characterized so far, and are summarized in Table 1, along with other known Ser/Thr sites of phosphorylation. Presence and Site(s) of Phosphorylation Determined by Mass Spectrometry. Mass spectrometry (MS) is the analytical method of choice for the characterization of protein phosphorylation.1,11 Matrix-assisted laser desorption ionization timeof-flight (MALDI ToF) mass spectrometric analysis of proteolytic peptides, before and after phosphatase treatment, identifies phosphopeptides by observation of an 80 Da mass reduction (HPO3).12,13 Although removal of the phosphate group establishes the presence of phosphorylation; it is then no longer possible to establish the location of the modification by subsequent tandem mass spectrometric experiments. Various methods are based upon the detection of diagnostic marker ions (typically PO3-) formed on collision-induced dissociation (CID) of phosphopeptides. Several groups have employed alternating (high and low) electrospray ionization (ESI) skimmer potentials to form PO3- ions (m/z 79) in the source region of the mass spectrometer during liquid chromatography (LC) MS experiments.14,15 Peptides eluting simultaneously with the detected PO3- ions are considered candidate phosphopeptides; however, no direct connectivity between the product ion and the precursor ion can be inferred. Scanning of negative ionic precursors of PO3- with tandem-in-space instruments allows for unambiguous connectivity between precursor and product ions.16-20 The specific detection of tyrosine phosphorylated peptides during a scan of positive-ion precursors of m/z 216.04 has also been described.21 More recently, it has become possible to identify sites of serine and threonine phosphorylation through a scan of positive-ion precursors of m/z 122.06.22 In that approach, β-elimination and Michael addition reactions replace the phosphate moiety with a functional group that forms abundant specific fragment ions during positive-ion CID. 374
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Scanning of ionic precursors of such fragment(s) reveals originally phosphorylated peptides. Although precursor ion scanning has been widely used for phosphopeptide identification, the limited mass resolution of the precursor ion spectrum may preclude determination of the charge state (and therefore the mass) of a precursor ion. Sensitivity, specificity, and dynamic range are improved by use of immobilized metal affinity chromatography (IMAC)23 to achieve selective isolation of phosphopeptides from a digest mixture prior to mass analysis.23-28 However, IMAC methods require multiple liquid handling steps (washing, binding of metal ion, etc.), and phosphopeptides elute from the columns either in base or phosphate buffer, requiring further clean up of samples prior to analysis. Thus, a number of alternative methods for the specific isolation of peptides following beta-elimination of the phosphate moiety, chemical modification(s), and the affinity isolation of the derivatized peptides have recently been introduced.29-31 However, multiple steps of derivatization require careful control of all conditions to maximize the yield at each step (essentially quantitative conversion at each step is essential if sensitivity is to be maintained) and to avoid false positives. Capillary LC/MS for Analysis of Phosphorylation. The low flow rate and high chromatographic resolution of capillary LC32 match the concentration range of microelectrospray33 mass spectrometry, by concentrating peptides into tens of nanoliters,34,35 rather than the microliter amounts required for analysis by direct infusion electrospray. Capillary LC-compatible methods for the identification of phosphopeptides are therefore desirable, e.g., identification of phosphopeptides through neutral loss of H3PO4 during collision-induced dissociation (CID), followed by a data-directed product ion analysis of the phosphorylated precursor ions.36 Similarly, observation of the loss of H3PO4 from [M - nH]n- peptide ions during infrared multiphoton dissociation (IRMPD) has also been described, but only for a direct infusion experiment.37 An ideal LC experiment would provide tandem mass spectrometric data from every peptide eluting from the column. Each peptide could then be identified from database searches, including any post-translational modifications. Unfortunately, data-dependent LC MS/MS experiments typically select only the few most abundant ions in any spectrum, thereby severely limiting the duty cycle, and biasing the results in favor of ions producing the strongest signals (due to higher abundance and/ or higher ionization efficiency), whereas low-abundance peptides may often be of higher interest. Obtaining product ion data from low-abundance peptides not immediately selected for product ion analysis is challenging, because components observed at low signal-to-noise ratio during capillary LC ESI MS experiments are rarely amenable to direct infusion ESI tandem mass spectrometry. An alternative approach is to identify (modified) peptides based upon mass alone. Peptide assignment then depends much less on signal magnitude, so that minor components may be identified as readily as more abundant peptides. Moreover, serine, threonine, and tyrosine phosphorylation can all be detected in the same experiment (without the need for chemical derivatization or affinity isolation). Indeed, any modification of known mass may be identified (e.g., acetylation, oxidation). Fourier Transform Ion Cyclotron Resonance (FT-ICR) MS. FT-ICR MS38 provides the highest available mass resolution, mass resolving power, and mass accuracy of any mass spectrometer (typically between 10 and 100 times greater than those
Characterization of Protein Kinase C Phosphorylation
achievable with other mass analyzers).39,40 ESI FT-ICR MS41,42 is therefore attractive for mass-based peptide identification. Although mass data alone cannot necessarily identify peptides from an entire protein database, peptide assignments can be made confidently at a mass accuracy of better than 5 ppm for a database limited to a few hundred proteins or less (as in this case).43,44 LC ESI FT-ICR performance may be optimized in several ways. (1) Several FT-ICR MS figures of merit (e.g., mass resolution and accuracy, dynamic range) independently improve at increasing magnetic field strength.45 (2) Ion cyclotron frequencies can shift according to the number of ions held in the Penning ion trap. If, as for LC/MS, the total number of ions varies significantly from scan to scan, internal calibration (i.e., conversion from measured ICR frequency to ion mass-tocharge ratio, m/z) is essential to allow low (and sub) ppm mass accuracy. (3) The time required to acquire the time-domain ICR signal and transfer the digitized signal to the control computer in a high-resolution FT-ICR MS experiment may be several seconds. External ion accumulation is therefore necessary to achieve an efficient experimental duty cycle. In this paper, we show how to identify (modified) peptides present at low stoichiometric ratios in complex mixtures. A vented capillary LC apparatus (see below) provides high chromatographic resolution (and therefore sensitivity), and our 9.4 T Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer provides the highest mass accuracy and resolving power of any current mass analyzer.46 External ion accumulation in a linear octopole ion trap allows the duty cycle of the experiment to be maximized. The introduction of calibrant ions of known m/z ratios into that trap allows for internal calibration of each mass spectrum. To introduce calibrant ions, with minimal reduction in experimental duty cycle, we constructed a dual ESI source capable of switching rapidly between two independent ESI emitters (only 400 ms is required to switch from one emitter to the other and back again). The LC ESI FTICR MS approach has been successfully applied to the identification of novel phosphorylated peptides generated from ingel tryptic hydrolysis of PKCR, PKCδ, and PKCζ. Modification of this method allows for observation of the neutral loss of H3PO4 from phosphopeptides during LC ESI FT-ICR MS experiments by incorporation of a 200 ms infrared multiphoton dissociation (IRMPD) event in alternate scans.
Experimental Section Expression, Isolation, and Digestion of PKC. Mouse PKC isotypes R, δ, and ζ were expressed as GST-fusion proteins in insect Sf-9 cells as previously described.47 The proteins were purified as described by use of GSSH-Sepharose.48 Thrombin cleavage (to obtain the enzyme without the GST tag) was performed with the protein still bound to the beads. Cleaved protein was eluted from the column and loaded onto SDSPAGE. The gel was stained with Coomassie Brillant Blue, and the prominent PKC band was excised and digested with trypsin as follows. Gel bands were subjected to multiple rounds of dehydration (15 min with 4:1 CH3CN/25 mM NH4HCO3 containing 0.1% formic acid) and rehydration (20 min with 25 mM NH4HCO3) until all of the Coomassie stain was removed. Following an additional dehydration step, the gel piece was rehydrated with 25 mM NH4HCO3 containing 10 mM dithiothreitol (DTT) and held at 37 °C for 45 min. After removal of excess DTT solution, 50 µL of a 25 mM NH4HCO3 solution containing 50 mM iodoacetamide (or iodoacetic acid) was added, and the reaction was allowed to proceed in the absence
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Figure 1. Top: Schematic representation of the dual electrospray ionization source. Bottom: “Blank” LC ESI FT-ICR mass spectrum showing the low magnitude of the calibrant ion signals.
of light for 45 min. Supernatant was then removed, and the gel piece was dehydrated, dried in a vacuum centrifuge, and rehydrated with a minimal volume of a 12.5 ng µL-1 solution of trypsin (Sequencing grade, Promega, Madison, WI) made up in 25 mM NH4HCO3. Digestion was allowed to proceed at 37 °C overnight. Prior to nanoelectrospray (nano-ESI) analysis,49 samples were desalted with a C18 ZipTip (Millipore, Billerica, MA) into 5 µL of a 4:1 CH3CN/H2O solution containing 0.1% formic acid, and electrosprayed directly from metal-coated nano-ESI needles (Protana, Odense, Denmark). Dual ESI Source. Our dual ESI source is similar to that originally introduced by Hannis and Muddiman.50 In our design (Figure 1, top) each microspray emitter is mounted on its own compact, 9.5 mm travel, three-axis dovetail stage (Newport Corp., Irvine, CA). One advantage of this design is the independent fine adjustment of the position of each emitter, with respect to the entrance of the ESI source capillary. This entire assembly is supported by two crossed roller bearing slides (DelTron Precision, Bethel, CT) that provide the range of motion required for alternately positioning each emitter in front of the ESI source capillary. One of those slides incorporates a pair of low-friction air cylinders that controls the x-position of the assembly. Compressed air (∼0.3 bar) is supplied through a fourway solenoid valve driven by an electrical circuit under control of the data acquisition system. Adjustable stops limit movement in each direction, and the maximum travel permitted by the slides is 25 mm. To preserve a stable electrospray from both emitters at all times, an extension plate (along the x axis) for the heated metal capillary entrance was constructed. It maintains a uniform electric field between the tip of each ESI emitter and the counter electrode, independent of x-position. For introduction of the calibrant ions into the storage octopole, a 10 pmol µL-1 mixture of two peptides (bradykinin and neurotensin, 1:1 CH3CN/H2O containing 0.1% formic acid) was electrosprayed from a 50 µM i.d. fused silica ESI emitter (400 nL min-1). Liquid Chromatography. Capillary LC was performed with a vented system similar in design to that described by Licklider Journal of Proteome Research • Vol. 2, No. 4, 2003 375
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vacuum interface53 and externally accumulated within a linear octopole trap.54 External accumulation was maintained during the entire experimental event sequence except during ion transfer (1.4 ms) to the open cylindrical ICR cell in which ions were captured by gated trapping. Ions were subjected to chirp excitation55,56 (48-480 kHz at 150 Hz/µs) and direct-mode broadband detection (1 M time-domain data). The total duration of a single scan was approximately 5 s, for an experimental duty cycle of >90% (based upon a down period of 400 ms to switch between ESI sources). Hanning apodization and one zero-fill were applied to all data prior to fast Fourier transformation and magnitude calculation.57 Frequency spectra were then calibrated58,59 internally from the measured ICR frequencies of bradykinin [M + 2H]2+ and neurotensin [M + 2H]2+ and [M + 3H]3+ (m/z 530.7879, 558.3105, and 836.9620).
Figure 2. Top: Total ion chromatogram (TIC) from LC ESI FTICR (9.4 T) analysis of 5 fmol each of bradykinin (BK) and neurotensin (NT). Peptides (1 fmol µL-1) were loaded onto the head of the column in artificial cerebrospinal fluid (acsf). Insets show an expansion from the spectrum obtained at each elution profile apex. Bottom: Schematic representation of the “vented” capillary LC apparatus, showing relative positions of the LC ESI emitter and the calibrant ESI emitter within the dual ESI source.
et al.35 and is illustrated in Figure 2, bottom. The 40 µL min-1 flow rate from the LC pump (Shimadzu 10AD, Shimadzu Scientific Instruments, Columbia, MD) was reduced to 1 µL min-1 with a home-built flow splitter prior to delivery to the injection valve (Rheodyne, Rhonert Park, CA). From the injection valve exit port, a 1 cm × 75 µm precolumn (Integra frit, BioBasic C18, New Objective Inc., Woburn, MA) was coupled to a 5 cm × 75 µm analytical column (Picofrit BioBasic C18, New Objective Inc.) through a low dead volume stainless steel T-piece (Valco Instruments Co., Inc., Houston, TX). The third port contained a 20 cm × 75 µm fused silica vent line. Samples (5 µL injected volume) were loaded onto the precolumn and desalted with the vent line open to waste at a flow rate of approximately 1 µL min-1. After 6 min, the vent line was closed, and flow was diverted with a reduced flow rate of 200 nL min-1 through the analytical column. Flow rate reduction was due to the increase in back pressure from the increased stationary phase bed length that reduced the ratio of the flow splitter; no change in the pump flow rate was required. Peptides were then eluted with a 6 min linear gradient of 100% A to 20% A and electrosprayed directly from the analytical column tip (15 µm i.d, A ) 2% CH3CN containing 0.05% formic acid; B ) 90% CH3CN containing 0.05% formic acid), thereby eliminating postcolumn dead volume and minimizing band broadening.33 The 2 kV potential required to initiate electrospray was applied directly to the T-piece between the precolumn and the analytical column. 9.4 T FT-ICR MS. Positive-ion experiments were performed with a home-built 9.4 T FT-ICR mass spectrometer51 under the control of a modular ICR data acquisition system (MIDAS).52 Ions were transported through a Chait-style atmosphere-to376
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7 T FT-ICR MS. Experiments were performed with a homebuilt 7 T FT-ICR instrument60,61 operating under the control of a MIDAS data station.52,62 Positive ions were transported through a Chait-style atmosphere-to-vacuum interface53 and externally accumulated within a linear octopole trap, modified to allow improved ion ejection along the z-axis.63 External accumulation54 was performed during the entire experimental event sequence except during the transfer (1.2 ms) to an open rectangular ICR cell, where ions were captured by gated trapping. Ions were subjected to chirp excitation (54-350 kHz at 150 Hz/µs) and direct-mode broadband detection (256 k data points). Hanning apodization and one zero fill were applied prior to fast Fourier transformation and magnitude calculation. Negative-ion nano ESI experiments were performed as for positive-ion measurements, with appropriate voltages reversed. FT-ICR frequency spectra were calibrated58,59 externally from the measured ICR frequencies of calibrant mixtures (Agilent Technologies, Wilmington, DE). Quadrupole MS. Nanoelectrospray experiments were performed with a Micromass Quattro Ultima instrument (Waters, Manchester, UK) operating in negative-ion mode. Scan range was 200 < m/z < 2000 over an 8 s period. LC ESI (Alternating IRMPD) FT-ICR MS and MS/MS. All LC experiments were performed with the 7 T FT-ICR instrument. A simple logic circuit enabled infrared irradiation from an external CO2 laser (10.6 µm, Synrad, Mukilteo, WA) of ions accumulated on alternate injections and transferred to the ICR cell. The circuit was placed between the MIDAS cell controller and the laser control box, and only every other transistor true logic (TTL) trigger received by the logic circuit was sent to the laser. Within the MIDAS experimental script, the irradiation period was 200 ms and the laser power was 18% of full power, or ∼7.2 W. Data Processing. Monoisotopic mass lists were generated for each spectrum by the THRASH algorithm.64 PKC peptides were identified by combining mass lists from 3 to 10 spectra into batches for Mascot65 (Matrix Science, London, UK) to be searched against the NCBInr database (mass tolerance ( 50 ppm). To ensure that PKC would be returned as the top hit, masses of expected PKC tryptic peptides were added to each mass list. Phosphorylation and methionine oxidation were selected as variable modifications. Monoisotopic masses (or m/z ratios) were calculated by use of IsoPro version 3.1 (MS/ MS Software, http://members.aol.com/msmssoft/). For LC ESI (alternate IRMPD) FT-ICR MS experiments, only spectra obtained with no IRMPD event were searched against the database.
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Results and Discussion Evaluation of the Dual ESI Source. Our dual ESI source was designed to switch rapidly between two independently operated ESI emitters spraying different samples (i.e., LC eluent or calibrant solution), as shown schematically in Figure 1 (top). To maximize the duty cycle of the LC ESI FT-ICR experiment, the period for introduction of calibrant ions was minimized. In normal operation, the length of time for the dual ESI source to switch emitters, introduce calibrant ions, and return to its original position was ∼400 ms. At that switching frequency, the calibrant electrospray emitter resided in front of the heated metal capillary for ∼50 ms. Concentrated (10 pmol µL-1) calibrant solutions sufficed to introduce a sufficient (small) number of ions into the accumulation octopole. (If the total ion population (and therefore charge density) within an accumulation multipole becomes too great, fragmentation, charge reduction, or ion ejection can occur.66,67 Initially, Agilent ESI calibrant solution provided ions spanning an appropriately wide m/z range. However, a calibrant mixture of two peptides (bradykinin and neurotensin at 10 pmol µL-1 each) provided a considerably cleaner “blank” spectrum with much less charge deposited into the accumulation octopole (and ultimately the Penning ion trap) (Figure 1, bottom). Mass accuracy typically deteriorates somewhat for peptide ions outside the m/z range spanned by the internal calibrant ions. However, it was deemed more important to keep the total charge contribution of the calibrant ions to the total ion population as low as possible to avoid any deleterious space-charge related effects. Evaluation of the Vented LC ESI FT-ICR Experiment. To obtain the maximum possible chromatographic resolution (and therefore sensitivity), peptides should be loaded directly onto the head of a capillary chromatography column and eluted with a short linear gradient. For simple mixtures (such as tryptic hydrolysates of a small number of proteins, as typically excised from a one- or two-dimensional gel), a 6-min gradient (200 nL min-1 flow rate) showed acceptable chromatographic resolution and maximum possible sensitivity. Any coeluting peptides are easily resolved by 9.4 T FT-ICR MS (data not shown). To demonstrate the sensitivity of this fast gradient approach, 5 µL of a 1 fmol µL-1 (5 fmol of each peptide) solution of bradykinin (RPPGFSPFR) and neurotensin (pGLYENKPRRPYIL) was loaded onto the head of a 5 cm × 75 µm C18 column at 200 nL min-1. To mimic a biological sample matrix, the peptides were dissolved in pure water (1 mg mL-1) and diluted to the required concentration with artificial cerebrospinal fluid (aCSF, 5 mM KCl containing 120 mM NaCl, 1.2 mM MgCl2, 1.8 mM CaCl2, 0.15% phosphate-buffered saline, pH 7.4). Figure 2 (top) clearly shows that both peptides elute from the column and are detected with good signal-to-noise (S/N) ratios within 15 min of the start of the gradient. The drawback of this type of LC is the significant length of time required to load each sample (for the experiment shown in Figure 2 (top), sample loading and desalting took 30 min). A vented column approach35 allows sample to be loaded onto a small precolumn at much higher flow rate, thereby saving tens of minutes if large sample volumes are required. Our capillary LC method therefore includes a precolumn and vent line. The design is based upon the principle described by Licklider et al.,35 but is constructed from commercially available columns, and no self-packing of stationary phase is required. Although subsequent experiments showed somewhat reduced chromatographic performance, sensitivity was not significantly reduced. In combination with the dual ESI source, 50 fmol of
Figure 3. Top: Mass spectrum obtained from scan 134 of LC ESI FT-ICR MS (9.4 T) analysis of PKCR tryptic hydrolysate. Following internal calibration, the [L240SVEIWDWDRTTR252 + HPO3 + 2H]2+ phosphopeptide ion was mass measured with an accuracy of +0.5 ppm (+0.0004 Th). Middle: Mass spectrum obtained from scan 90 of the LC ESI FT-ICR MS (9.4 T) analysis of PKCR tryptic hydrolysate. * ) calibrant ions. Both the phosphorylated and nonphosphorylated analogues of the V317ISPSEDR324 peptide are seen. The experimental mass difference between the phosphorylated and nonphosphorylated forms ([M + H]+ ions) was 79.9686 + 0.0023 Da (HPO3 ) 79.9663 Da). Bottom: Expanded segment of a negative-ion direct infusion nano-ESI FT-ICR (7 T) mass spectrum of a PKCR tryptic hydrolysate. Both the singly and doubly phosphorylated forms of the T87 peptide (residues 633-672) are observed. Following external calibration, the [M + (HPO3) - 3H]3- and [M + HPO3)2 - 3H]3masses were measured to within -12.2 and +56 ppm. The measured mass difference (between the most abundant isotopic peaks) was HPO3 + 0.074 Da. Observation of those peptides confirms two of the three known sites of PKCR phosphorylation.
standard peptides could be detected reproducibly at good S/N ratio. All samples contained the three internal calibrant ions shown in Figure 1 (bottom). The total experiment, including loading, desalting, gradient elution, and reequilibration of the columns, took ∼35 min. The vented capillary LC apparatus illustrated in Figure 2 (bottom) was employed for all subsequent experiments. PKCr. A single Coomassie brilliant blue-stained PKCR gel spot was excised from a 1D gel and subjected to in-gel tryptic hydrolysis. Five microliters of the digest supernatant was then analyzed with the vented LC ESI FT-ICR method. Forty-seven peptide ions were detected, including two phosphopeptides. The detected peptides, along with the errors in mass measurement, are detailed in Table 2. Note that the absolute mean of the measured mass errors (from 47 peptides) decreased from 11.5 to 1.5 ppm when spectra were internally calibrated. The detected peptides span 41% of the PKCR primary amino acid sequence. The doubly protonated, phosphorylated peptide ion (L240SVEIWDWDRTTR252) was detected in scan 134 (Figure 3, top). Following internal calibration, the mass measurement error was +0.5 ppm (+0.0004 Th or +0.0008 Da). Of the three Journal of Proteome Research • Vol. 2, No. 4, 2003 377
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Table 2. Peptides Detected during a Single Capillary LC ESI FT-ICR MS Analysis of a PKCR Tryptic Hydrolysatea assignment
sequence
calcd mass (m)
(z)
measured m/z
calcd m/z
error (ppm)
78-99 + (Cmc)2 166-178 173-178 173-178 182-197 200-205 231-238 239-249 240-249 240-252 + HPO3 253-268 253-268 + O 269-276 269-276 269-276 + O 317-324 317-324 317-324 + HPO3 325-333 336-347 336-347 + O 353-358 353-358 + O 359-368 360-368 390-412 + Cmc 458-462 463-478 463-478 + O 463-478 + O 479-486 + Cmc 487-496 487-496 + O 487-496 + (O)2 497-517 + Cmc 564-570 + Cmc 581-589 + Cmc 581-592 + Cmc 593-598 593-598 599-604 599-608 600-604 609-617 622-628 622-632 629-632
CHEFVTFSCPGADKGPDTDDPR AEVTDEKLHVTVR LHVTVR LHVTVR NLIPMDPNGLSDPYVK LIPDPK LKPSDKDR RLSVEIWDWDR LSVEIWDWDR LSVEIWDWDRTTR NDFMGSLSFGVSELMK NDFMGSLSFGVSELMK MPASGWYK MPASGWYK MPASGWYK VISPSEDR VISPSEDR VISPSEDR KQPSNNLDR LTDFNFLMVLGK LTDFNFLMVLGK VMLADR VMLADR KGTEELYAIK GTEELYAIK VLALLDKPPFLTQLHSCFQTVDR GIIYR DLKLDNVMLDSEGHIK DLKLDNVMLDSEGHIK DLKLDNVMLDSEGHIK IADFGMCK EHMMDGVTTR EHMMDGVTTR EHMMDGVTTR TFCGTPDYIAPEIIAYQPYGK EAVSICK LGCGPEGER LGCGPEGERDVR EHAFFR EHAFFR RIDWEK RIDWEKLENR IDWEK EIQPPFKPK GAENFDK GAENFDKFFTR FFTR
2509.0161 1495.7994 723.4391 723.4391 1771.8815 681.4061 957.5243 1473.7365 1317.6353 1755.7981 1760.8113 1776.8062 938.4320 938.4320 954.4269 901.4504 901.4504 981.4167 1070.5469 1396.7424 1412.7373 703.3687 719.3636 1150.6234 1022.5284 2698.4153 620.3646 1825.9242 1841.9192 1841.9192 941.3987 1175.5062 1191.5011 1207.4961 2404.1296 806.3843 974.4127 1344.6091 805.3871 805.3871 845.4395 1357.7102 689.3384 1082.6124 779.3450 1330.6305 569.2961
(3) (2) (1) (2) (2) (1) (2) (2) (2) (2) (2) (2) (2) (1) (1) (2) (1) (1) (2) (2) (2) (1) (1) (2) (1) (3) (1) (2) (3) (2) (1) (2) (2) (2) (2) (1) (1) (2) (1) (2) (2) (2) (1) (2) (1) (2) (1)
837.3508 748.9074 724.4474 362.7269 886.9505 682.4104 479.7695 737.8751 659.8226 878.9067 881.4104 889.4089 470.2223 939.4371 955.4339 451.7327 902.4575 982.4261 536.281 699.3783 707.3759 704.3766 720.3718 576.3187 1023.5384 900.4808 621.3710 913.9673 614.9808 921.9650 942.4102 588.7612 596.7580 604.7560 1203.0721 807.3923 975.4203 673.3113 806.3947 403.7002 423.7272 679.8635 690.3461 542.3135 780.3534 666.3228 570.303
837.3460 748.9070 724.4464 362.7268 886.9480 682.4134 479.7694 737.8755 659.8249 878.9063 881.4129 889.4104 470.2233 939.4393 955.4342 451.7325 902.4577 982.4241 536.2807 699.3785 707.3759 704.3759 720.3709 576.3189 1023.5357 900.4790 621.3718 913.9694 614.9803 921.9669 942.4059 588.7604 596.7579 604.7553 1203.0721 807.3917 975.4200 673.3118 806.3944 403.7008 423.7270 679.8624 690.3457 542.3135 780.3522 666.3226 570.3034
5.7 0.5 1.4 0.3 2.8 -4.4 0.2 -0.5 -3.5 0.5 -2.8 -1.7 -2.1 -2.3 -0.3 0.4 -0.2 2.0 0.6 -0.3 0.0 1.0 1.2 -0.3 2.6 2.0 -1.3 -2.3 0.8 -2.1 4.6 1.4 0.2 1.2 5.4 0.7 0.3 -0.7 0.4 -1.5 0.5 1.6 0.6 0.0 1.5 0.3 -0.7
(9.5) (|11.5|)
0.2 |1.5| 23/47 41%
(values in parentheses were calculated without internal calibration)
a
Data represent masses obtained from individual MS spectra. Cmc ) carboxymethylcysteine.
potential phosphorylation sites in that peptide, T250 has previously been identified as an autophosphorylation site of PKCR.68 In scan 90 (Figure 3, middle), both the phosphorylated (+2 ppm) and nonphosphorylated (-0.2 ppm) forms of V317ISPSEDR324 are observed. That sequence lies within the V3 or hinge region of PKCR. Initially, that region was thought to allow for flexibility of the enzyme during activation. Furthermore, proteolytic degradation after activation starts in the hinge region. Recently, the importance of the region in intracellular distribution of PKCR has been demonstrated.69 It is therefore likely that its phosphorylation might influence PKCR targeting. The experimental mass difference between the phosphorylated and nonphosphorylated [M + H]+ ions was 79.9686 Da (i.e., 0.0023 Da higher than the 79.9663 mass of HPO3). In fact, the only possible elemental composition, based on combination of H, C, O, N, P, and S, for 79.9663 ( 0.0023 Da is HPO3. Thus, 378
mean |mean|
