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Quantification of acetylation at proximal lysine residues using isotopic labeling and tandem mass spectrometry
Methods 36 (2005) 395–403 www.elsevier.com/locate/ymeth
QuantiWcation of acetylation at proximal lysine residues using isotopic labeling and tandem mass spectrometry Christine M. Smith ¤ Department of Chemistry, University of Puget Sound, 1500 N. Warner, Tacoma, WA, USA Accepted 30 March 2005
Abstract With the emergence of the histone code as a key determinant in the regulation of gene expression, it has been important to develop tools that can not only identify the types and locations of myriad modiWcations, but also determine how the levels of these modiWcations change as a result of various processes in a cell. Mass spectrometry has become a method of choice for the investigation of post-translational modiWcations in histone proteins. Described in this article is a mass spectrometric method that is useful for direct quantiWcation of levels of acetylation at lysines residues in close proximity to one another, as is the case for the amino terminal tail of histone H4. This method involves fragmentation of peptides into b and y ions that contain one or more sites of modiWcation and isotopic labeling which ensures equivalent ionization and fragmentation. 2005 Elsevier Inc. All rights reserved.
1. Introduction Mass spectrometry (MS) has become a leading method for the proteomic analysis of post-translational modiWcations [1–4]. Extending beyond MS analyses that determine the location of modiWcations, the method presented here provides information on how to quantify the level of modiWcation at a particular amino acid residue. MS analyses commonly involve proteolytic digestion of a mixture of proteins into numerous peptides. The peptides can be directly ionized by matrix-assisted laser desorption ionization (MALDI) or electrospray ionization (ESI), or ionized by either of these methods following on-line (ESI) or oV-line (MALDI or ESI) chromatographic separation by reverse-phase liquid chromatography (LC). The presence of modiWcations can be inferred from diVerences in the mass-to-charge ratios (m/z) observed for modiWed and unmodiWed amino acid residues. Peptides containing a single site of modiWcation can be analyzed by MS using parent ions, *
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which are ions carrying an extra proton (MH+). However, if more than one site of modiWcation is present within a peptide, tandem mass spectrometry (MS/MS) is required. In an MS/MS experiment, the peptide backbone is fragmented in a process called collision-induced dissociation (CID). Bond breakage occurs predominantly at amide bonds (usually at one site per molecule) creating a mixed population of N-terminal and C-terminal ions, which are referred to as b and y ions, respectively. (A useful tutorial can be found in [5].) For mass spectrometry to be used quantitatively, it is important that proteolysis, ionization, and fragmentation occur equivalently for both modiWed and unmodiWed peptides. One way to ensure equivalency is to use isotopic labeling which creates a chemically identical population of molecules that can be distinguished by mass. The method described in this article combines CID MS/MS and isotopic labeling to directly measure endogenous levels of acetylation at individual lysine residues. This method was initially used to compare the level of acetylation between wild-type histone H4 and H4 mutants that increased telomeric silencing [6]. The data presented below come from the analysis of an H4
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C.M. Smith / Methods 36 (2005) 395–403
mutant that has a G7 ! I mutation (Fig. 1A). This mutant (called H4_G7I) was isolated from yeast cells using a procedure established by Waterborg [7]. Since histone proteins isolated from cells are variably acetylated, it was necessary to create a chemically identical population of histones that could be used in a quantitative analysis. This was accomplished by acetylating, in vitro, all unmodiWed lysine residues with deuterated acetic anhydride. Since the mass diVerence between a protiated acetyl group (42 Da) and a deuterated acetyl group (45 Da) is 3 Da, it was possible to distinguish by mass spectrometry between lysine residues that were acetylated in vivo and those that were acetylated in vitro. A schematic showing the acetylation procedure for histone H4 is presented in Fig. 1B. Following chemical acetylation, H4_G7I was digested with the protease trypsin. Normally trypsin cleaves the peptide backbone following lysine and arginine residues. However, the presence of acetyl groups on lysine residues prevents tryptic cleavage at these sites. The four lysine residues within the amino terminal tail of histone H4 were thus encompassed in a single proteolytic fragment containing residues 4–17, abbreviated as H4_G7I(4–17). One of the challenges in quantifying acetylation within the amino terminal tails of histones is the abundance of lysine residues in close proximity to one another. For this reason, CID-MS/MS was required to fragment the parent ion into smaller b and y ions containing one, two or three acetylated sites. The 3 Da diVerences in mass observed for both parent and fragment ions allowed us to determine the levels of acetylation at individual lysine residues. To understand how data from MS and MS/MS analyses provided information about the acetylation levels of ions containing more than one acetylated lysine residue, consider that an ion being analyzed contains n modiWed residues. Each of the residues can be in one of two states: H represents the presence of a protiated acetyl and D represents the presence of a deuterated acetyl. There are only two possible states for an ion with only one modiWed residue (H or D) and therefore only two clusters of m/z peaks in the mass spectrum of such ions. (Each cluster has multiple peaks due to the natural abundance of heavy isotopes such as 13 C, 15N, and 18O. The term ‘monoisotopic’ is used below to describe the Wrst peak in the cluster.) To determine the level of in vivo acetylation at this particular site, one need only determine the ratio of H to the total amount of H and D, that is H/(H + D). This simple analysis is not possible for an ion containing more than one modiWed residue. For an ion with two modiWed residues, there are four possible states: HH, HD, DH, and DD; however, HD and DH will have the same mass so they are indistinguishable by MS. Thus, in the mass spectrum of such an ion, there are three monoisotopic m/z peaks. Extending this further, for an ion with three modiWed residues, there are eight possible states (HHH, HHD, HDH,
DHH, DDH, DHD, HDD, and DDD) of which two groups of three have the same mass (HHD, HDH, and DHH and DDH, DHD, and HDD), resulting in four monoisotopic m/z peaks. Similarly, for an ion with four modiWed residues, there are 12 possible states, resulting in Wve m/z peaks. Thus, for an ion with n modiWed residues, there will be n + 1 monoisotopic m/z peaks in the mass spectrum of that ion. Mathematical expressions, showing how the level of acetylation in parent and fragment ions containing multiple modiWed residues can be determined from other ions with fewer modiWed residues, are described in detail in Smith et al. [8]. In this previous work, we showed that the fraction of in vivo acetylation at a particular site can be determined by summing up all of the in vivo acetylations (normalized) within a given CID fragment and subtracting from this the fraction of in vivo acetylation at all sites other than the one of interest. In essence, the total number of in vivo acetylations within a given fragment was determined by “weighting” each of the peaks according to the number of protium acetylations represented by that peak. This sum was then normalized by dividing by the sum of the intensities for all species (HHHH through DDDD) and subtracting from the total the fraction of acetylation at each site for which the fraction of acetylation had been determined using other types of CID fragment ions. Since an in-depth treatment of theory behind the quantitative aspects of this method has been published elsewhere [8], the goal of this article is to serve as a more practical guide for carrying out this method.
2. Description of method 2.1. List of materials 1. Proteins puriWed by reversed-phase HPLC (see description below). 2. Deuterated acetic acid (Acros, #166210050) and deuterated acetic anhydride (Acros, #174670050). 3. TPCK-treated trypsin (Worthington, #LS003740) and ammonium bicarbonate (Sigma, #A6141). 4. LC electrospray ionization ion trap mass spectrometer. (We have used ThermoFinnigan LCQ instruments at Fred Hutchinson Cancer Research Center in Seattle, WA; the Nebraska Center for Mass spectrometry in Lincoln, NE; and the laboratory of Dr. Larry David at Oregon Health and Science University using both electrospray and MALDI (MassTech, Columbia, MD) sources.) 5. ThermoFinnigan Xcalibur and Microsoft Excel software. 6. HPLC grade acetonitrile (Fisher, #A998-4) with 0.1% triXuoroacetic acid (Sigma–Aldrich, #T62200) or 0.2% acetic acid (Fisher, A38-212).
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Fig. 1. Flowchart describing the experimental procedure used to quantify site speciWc levels of histone H4 acetylation. (A) Amino terminal tail region of the histone H4_G7I mutant containing four lysine residues, located at positions 5, 8, 12, and 16. In most species the N-terminus is blocked by acetylation. The arrows above the sequence indicate sites of tryptic digestion (following complete acetylation of histone H4, see below) that generate the H4_G7I(4–17) peptide. (B) Schematized population of histone H4 molecules; open circles indicate the presence of an in vivo acetylation. Following HPLC puriWcation, the histone H4 proteins are treated with deuterated acetic anhydride to acetylate all lysine residues not acetylated endogenously; the closed circles indicate the presence of a deuterated acetyl moiety. Treatment with trypsin results in cleavage following arginine (R) 3 and R17 (see (A)), producing a chemically identical population of histone H4_G7I peptides, containing residues 4–17, for which the lysine residues acetylated in vivo are tagged with a protiated acetyl group and those that were acetylated in vitro are tagged with a deuterated acetyl group. Since there is a 3 Da mass diVerence between protiated and deuterated acetyl groups (42 Da versus 45 Da, respectively), the endogenous level of acetylation can be determined using mass spectrometry.
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7. Speed vacuum microfuge and temperature-controlled bath. 2.2. Obtaining puriWed histones The yeast histone puriWcation procedure established by Waterborg [7] has repeatedly produced excellent protein samples for MS analysis. A description of Waterborg’s puriWcation of histones from crude yeast nuclei can be found at http://www.fhcrc.org/labs/gottschling/ yeast/. Our only deviation from this procedure involved adding the deacetylase inhibitor zinc sulfate (at a Wnal concentration of 2 mM) to the nuclear isolation buVer. After isolating the Xow-through from centricon devices, histone containing fractions were combined in microfuge tubes and dried down to »100 L using a speed vacuum microfuge. These proteins were then injected onto a Zorbax 150 £ 4.6 mm C-18 column (Alltech, catalogue #883995-902) and eluted using a gradient that increased from 2 to 40% acetonitrile (ACN)/0.1% triXuoroacetic acid (TFA) over 10 min and then increased from 40 to 60% ACN/0.1% TFA over a 40-min period. The Xow rate was maintained at 1 mL/min throughout the separation. Under these conditions, histone H4 co-eluted with H2A at »43% ACN/0.1% TFA and histone H3 eluted »50% ACN/0.1% TFA. To be certain about the identity of these proteins when performing the puriWcation for the Wrst time, SDS–PAGE and Western blot analysis using antibodies to histones should be used (these antibodies can be purchased from Upstate). HPLC puriWed histone samples were then dried in microfuge tubes using a vacuum concentrator and stored at either 4 or ¡20 °C; the protein pellets were either clear or white. 2.3. In vitro acetylation PuriWed and dried histone samples were resuspended in 50 L deuterated acetic acid and 5 L deuterated acetic anhydride and left at room temperature for 6 h. Although this reaction is carried out under acidic conditions, it proceeds to completion because of the large change in free energy resulting from the conversion of an anhydride and amine to a carboxylic acid and amide. The acetic anhydride also acetylates amino acids containing hydroxyl residues; however, this occurs to a lesser extent than with amines. Nonetheless, one should be aware of any serine, threonine, and tyrosine residues in peptides being analyzed by this method. If this is the case, the esteriWed residues can be hydrolyzed under mild alkaline conditions, leaving only the acetylated lysines [9]. Prior to quantiWcation, the degree of acetylation should be ascertained. This can be accomplished by determining the mass of the whole (undigested) protein following the acetylation step. When a mutant form of histone H4 was acetylated and analyzed by mass spec-
trometry, we found six peaks diVering from one another by 42–45 Da; the largest peak occurred at the mass of the protein if all 11 lysine residues of histone H4 were acetylated (data not shown). The peaks representing masses higher than the fully acetylated protein were likely due to acetylation of serine and threonine residues (see above). Since there were no serine, threonine, and tyrosine residues within the amino terminal tail of H4_G7I(4–17), we did not carry out an alkaline hydrolysis step. 2.4. Trypsin digestion Following acetylation, samples were completely dried in a speed vacuum microfuge to evaporate the acetic acid and acetic anhydride, resuspended in 20 mM ammonium bicarbonate with 100 ng trypsin (in 50 L Wnal volume), and incubated at 37 °C overnight. Prior to MS analysis, the ammonium bicarbonate buVer was evaporated using the speed vacuum microfuge. Our MS analyses indicated a signiWcant level of acetylation at the newly formed N-terminus of the H4-G7I(4–17) typtic peptide (see Fig. 1A). This was likely the result of reaction of the amino group of Gly4 with residual acetic anhydride. To eliminate this problem, the acetic anhydride and acetic acid should be neutralized by the addition of mild base prior to evaporation and digestion with trypsin. 2.5. Mass spectrometry There are a number of ways that MS can be used to determine the masses of peptides containing protiated and deuterated acetyl groups. We have investigated both ESI and MALDI of our samples using the LCQ ion trap instrument. While both methods provided excellent MS data, we found that the singly charged ions produced by MALDI did not generate suYcient quality MS/MS spectra after fragmentation in the ion trap. What follows below is a description of the conditions successfully used to analyze the H4_G7I(4–17) peptide with on-line LC followed by ESI and ion trap CID MS/MS. Acetylated and digested histones (H2A and H4) from »500 mL of yeast culture were dissolved in »20 L of 5% formic acid; 2 L of this sample was injected, using an autosampler, onto an Agilent 1100 Series capillary LC system on-line with the ThermoFinnigan LCQ Classic mass spectrometer. In a mobile phase A consisting of 0.2% acetic acid, the sample was loaded onto a 2 cm £ 180 m trap cartridge containing 5 m particle size Zorbax SB-C18 resin (Agilent Technologies) at a Xow rate of 5 L/min. After 10 min of washing, the Xow rate was decreased to 1.5 L/min and the peptides were transferred (on-line) to a 10 cm £ 180 m column containing the same packing material as the trap cartridge. The fused silica of the column lead directly into the Wxed
C.M. Smith / Methods 36 (2005) 395–403
spray tip of the standard ESI source modiWed with a 34 G metal needle (ThermoFinnigan, Cat. No 9714420040). Both trap and column were slurry packed in a 360 m outer diameter fused silica capillary tubing terminated with a microWlter assembly (Upchurch ScientiWc, #M-520). Peptides were eluted with a mobile phase B containing 75% ACN and 0.2% acetic acid. The gradient increased from 2 to 10% B over 10 s and then gradually increased from 10% B to 35% B over 50 min. Under these conditions, the variable isotopic content of the H4 peptides only slightly aVected their elution times. The mass spectrometer began acquiring scans from m/ z 400–2000 (in proWle mode) 10 min after the gradient was started. Under the data-dependent settings, the mass spectrometer was conWgured to acquire MS/MS scans (also in proWle mode) of parent ions with m/z 750.9, which corresponds to the average m/z of the various protiated and deuterated forms of the doubly charged ion of the H4_G7I(4–17) peptide. To activate this feature, the Nth most intense ion from the list button, accessed from the scan event tab, must be activated and a value of 1 entered. The collision energy of CID fragmentation was set at 40% and an isolation mass window of 8 Da was used to isolate all of the isotopic variants—from peptides containing four protiated acetyls to peptides containing four deuterated acetyls. We found that under our condi-
399
tions, a single scan did not provide high enough signal to noise for quantiWcation measurements. However, this problem could be alleviated by averaging several scans. Thus, it is important that this type of mass spectrometer be programmed to perform MS/MS on only the peptide(s) of interest so that the maximum number of scans can be acquired as the peptide elutes from the column. This is accomplished by disabling the dynamic exclusion function of the software and assuring that the minimum MS signal for initiating an MS/MS scan is set suYciently low. It should be noted that oV-line separation followed by MALDI CID recording of the CID spectra might also provide improved signal-to-noise ratios since scans could be acquired on the entire sample. In this case, there would be no chromatographic peak width limitation. 2.6. Data analysis The Xcalibur software that comes standard with the ThermoFinnigan LCQ instrument was used to measure ion currents of parent ions (from MS spectra) and fragment ions (from MS/MS spectra), which in turn allowed us to determine the level of acetylation at various lysine residues. MS data Wles were displayed as two stacked windows containing a plot of the total ion current as a function of time (the chromatogram) on top of a plot of
Fig. 2. Chromatograms of histone H4 and H2A peptides eluting from the on-line LC column. (A) The relative abundance of histone H4 and H2A tryptic peptides, measured by ion current in the ion trap of the mass spectrometer, is plotted as a function of elution time from the on-line column. (B) Chromatogram showing the elution times of peptides within 1 Da of m/z 750.9. Viewing the mass spectrum of the peak at »20 min shows that this peak contains the H4_G7I(4–17) peptide (see Fig. 3).
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the ion current as a function of m/z (the spectrum). Fig. 2A shows the full chromatogram for the histone H2A and H4 mixture. Under the ‘ranges’ option in the chromatogram window, a ‘full ms’ scan Wlter and a ‘mass range’ at 750.9 were selected, showing the time at which peptides of this m/z eluted from the on-line column (Fig. 2B). With the spectrum window active, the mouse cursor was dragged across individual chromatogram peaks causing the averaged MS spectra of these
parent ions to be displayed along with other ions that eluted from the column at this time. The H4_G7I(4–17) peptide eluted »20 min after the MS analysis began; singly and doubly charged H4_G7I(4–17) peptides, in their Wve variably acetylated forms, were seen at m/z »1504 and m/z »752.5, respectively (Fig. 3A). The overall number of deuterated and protiated acetyls were observed by dragging the cursor across the singly charged parent ion (see expanded view in Fig. 3A). To view the averaged MS/
Fig. 3. LC-ESI spectra of isotopically labeled histone H4_G7I(4–17) tryptic peptide. (A) Mass spectrum showing the m/z and relative abundance of all species eluting with the histone H4_G7I(4–17). The most abundant m/z of singly (MH+) and doubly (MH2+) charged H4_G7I(4–17) ions are 1503.9 and 752.5 Da, respectively. Expanded above the spectrum are the clusters of peaks that correspond to the Wve possible protiated and deuterated forms of the fully acetylated isotopically labeled H4(4–17); 0D, 1D, 2D, etc. mark the peaks representing peptides with zero, one, two, etc. deuterated acetyl groups. (B) Tandem mass spectrum (MS/MS) showing the relative abundance of fragment ions of the H4_G7I(4–17). Peaks are labeled according to ion number for the b and y ions described in (C). In some cases, a loss of water caused the mass of the fragment ions to be 18 Da lower than expected (see b7 and b9). (C) Diagram of fully acetylated histone H4_G7I(4–17), showing the masses of b and y ions, indicated above and below the sequence, respectively. The masses represent fragments containing only protiated acetyl groups; each deuterated acetyl group increases the mass by 3 Da.
C.M. Smith / Methods 36 (2005) 395–403
MS scans of the 750.9 parent ion, the ‘ms/ms’ Wlter was selected under ‘ranges’ in the spectrum window (Fig. 3B). Close-up views of the peaks in the MS/MS spectrum were obtained by dragging the cursor across speciWc peaks (Fig. 4). This allowed the 3 Da mass diVerences of the various H4_G7I(4–17) fragments to be observed. As discussed in Section 1, ions containing n acetylated lysine residues produce n + 1 m/z peaks. Because of the natural abundance of heavy isotopes, each monoisotopic peak is followed by smaller isotope peaks, each at one a.m.u. higher m/z. The presence of both in vivo and in vitro acetylations within each fragment causes the monoisotopic peaks to diVer from one another in 3 Da increments. To measure the abundance of the various protiated and deuterated forms of a particular b, y or parent ion, ion currents (or intensities) of peaks were measured by dragging the cursor across each cluster of peaks (see Fig. 4A), with the chromatogram window active, and recording in an Excel Wle (see below) the normalized number displayed in the upper right corner of the chromatogram window. As mentioned earlier, under our conditions, the multiple MS/MS scans of the doubly charged peptide were needed to obtain the highest quality peak pattern. Fig. 4B demonstrates how averaging individual scans produced smooth peaks whose ion currents can be readily determined. Not every MS/MS fragment of the parent ion was suitable for quantiWcation, even after averaging; therefore, it was necessary to assess each cluster of peaks before carrying out the quantitation. However, obtaining a suYcient number of quality peaks was not a problem since there were multi-
401
ple b and y ions that could be used to analyze each lysine residue [6,8]. Ion currents of the peaks corresponding to the variably protiated and deuterated forms of b, y, and MH+ ions were entered into columns of an Excel spreadsheet (Table 1). The fraction of each of the variably protiated and deuterated species was determined by dividing the intensity of each species by the sum of the intensities of all the species for a particular ion. For example, for an ion with two lysine residues the fraction of ions with two protiated acetyls is HH/(HH + HD/DH + DD). A formula that executed this calculation was entered into the equation box (G) titled ‘0D’ since the HH ion contains no deuterated acetyls groups. To determine the fractional contribution various acetylated and deuterated species made to the total number of ions of a particular m/z, similar formulas were entered into boxes H, I, J, and K, which were titled ‘1D,’ ‘2D,’ ‘3D,’ and ‘4D.’ Next, the fraction of in vivo acetylation (fac) was determined. For fragments containing one lysine residue, fac is the same value as in the 0D column. For fragments containing more than one lysine residue, fac was determined by multiplying the values in the 0D through 3D columns by the number of protiated acetyls present in the ions they represent and subtracting from this sum the average fac for the other lysines within the fragment ion. Based on the example grid boxes in Table 1, for an ion consisting of two lysine residues the formula was ‘(2 ¤ G + 1 ¤ H) ¡ M2’; for an ion consisting of three lysine residues, the formula was ‘(3 ¤ G + 2 ¤ H + 1 ¤ I) ¡ (M2 + M3)’; and for an ion consisting of three lysine residues, the formula was
Fig. 4. Expanded mass spectra of MS/MS ions of H4_G7I(4–17). (A) Ion intensities were determined from selected ion plots for each isotope cluster (a monoisotopic peak and the following two isotope peaks). Masses of fragmentation ions are indicated. See Fig. 3C for the peptide sequence corresponding to each m/z. The ion current was measured by dragging the cursor across each clusters of three peaks (arrows) and then recording the normalized (NL) values from the chromatogram window (the numbers above each arrow) in Excel spreadsheet boxes (Table 1). (B) The top spectrum is the average of eight individual scans (below); this demonstrates the importance of averaging spectra to maximize peak quality.
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Table 1 Example Excel spreadsheet for determining the fraction of acetylation at lysines 5, 8, 12, and 16 of histone H4_G7I Aa 1y ionb 2y5(530) 3y7(757) 4y10(1097) 5MH+(1494) a b c d e
B
C
D
E
F
G
H
I
J
K
L
M
ICc 1.01E+06 2.08E+05 1.02E+05 2.6E+05
IC 3.03E+05 7.84E+05 5.00E+05 5.4E+05
IC
IC
IC
1D
2D
3D
4D
1.35E+05 5.75E+05 2.0E+06
1.11E+05 2.5E+06
0Dd 0.77 0.18 0.08 0.04
0.70 0.39 0.09
0.12 0.45 0.32
0.09 0.41
0.14
Lys residue 16 12 8 5
fAce 0.77 0.30 0.40 0.02
8.8E+05
Grid box names from Excel spreadsheet are correlated with description in text. y ions, m/z of C-terminal fragments ions. IC, ion current measurements for a particular peak, beginning with the lowest m/z of the set of peaks. 0D, 1D, 2D, 3D, and 4D, the fraction of ions containing zero, one, two, three or four deuterated acetyl groups, respectively. fAc, fraction of acetylation at individual lysine residues.
‘(4 ¤ G + 3 ¤ H + 2 ¤ I + 1 ¤ J) ¡ (M2 + M3 + M4),’ where the M column represents the fac of already determined lysine residues within a particular fragment. Since multiple ions were available for analysis, fac values were averaged and multiplied by 100 to give the average percent acetylation at each lysine residue. An example spreadsheet showing all of the data we acquired for the acetylation analysis of another histone H4 mutant (A15T) is available at http://chem-ncms.unl.edu/SupFile.html.
information presented here is intended to serve in conjunction with Smith et al. [8], which contains a detailed description of the theory behind the quantitation strategy for ions containing more than one site of modiWcation. While this method has focused on acetylation of histones, it could also serve as a guide for the quantiWcation of other modiWcations that are located near one another in the primary structure of a protein.
Acknowledgments 3. Concluding remarks Within the past few years, the number of publications describing the type and location of modiWcations in histones has increased dramatically. To catalogue these numerous modiWcations, researchers have used modiWcation speciWc fragmentation patterns [10], construction of histone modiWcation databases that can be searched using specialized computer programs [11], and high resolution mass spectrometers, such as quadrupole time-of-Xight and ESI Fourier-Transform ion cyclotron resonance instruments [12,13]. Quantitative MS analyses have also been used to investigate changes in the levels of histone H3 methylation and acetylation in mouse embryonic stem (ES) cells [14], yeast [15], and Drosophila Kc cells [16]. The ES cell studies involved the use of chemically synthesized and modiWed peptide standards to account for variability in ionization and fragmentation between samples [14]. The yeast and Drosophila studies by-passed the need for chemical equivalency by comparing the parent masses of modiWed peptides isolated from cells with diVerent genetic backgrounds [15,16]. The method described in this article focuses on the direct and accurate measurement of acetylation at individual lysine residues that are in close proximity to one another in a tryptic peptide, and it involves the use of a ThermoFinnigan LCQ mass spectrometer, which is one of the least costly and most commonly found mass spectrometers in laboratories today. This proteomic approach is diVerent from other quantitative MS approaches of histone modiWcation in that it combines isotopic labeling using deuterated acetic anhydride and CID MS/MS. The
A special thanks to Larry David and Zhongli Zhang for running the G7I mutant sample. Support for the analysis of samples at OHSU was provided by National Eye Institute Core Grant EY10572. Additional thanks go to Larry David and David and Jean Smith for helpful conversations. And my appreciation goes to the Gottschling lab for helping me gather information for this article.
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QuantiWcation of acetylation at proximal lysine residues using isotopic labeling and tandem mass spectrometry Christine M. Smith ¤ Department of Chemistry, University of Puget Sound, 1500 N. Warner, Tacoma, WA, USA Accepted 30 March 2005
Abstract With the emergence of the histone code as a key determinant in the regulation of gene expression, it has been important to develop tools that can not only identify the types and locations of myriad modiWcations, but also determine how the levels of these modiWcations change as a result of various processes in a cell. Mass spectrometry has become a method of choice for the investigation of post-translational modiWcations in histone proteins. Described in this article is a mass spectrometric method that is useful for direct quantiWcation of levels of acetylation at lysines residues in close proximity to one another, as is the case for the amino terminal tail of histone H4. This method involves fragmentation of peptides into b and y ions that contain one or more sites of modiWcation and isotopic labeling which ensures equivalent ionization and fragmentation. 2005 Elsevier Inc. All rights reserved.
1. Introduction Mass spectrometry (MS) has become a leading method for the proteomic analysis of post-translational modiWcations [1–4]. Extending beyond MS analyses that determine the location of modiWcations, the method presented here provides information on how to quantify the level of modiWcation at a particular amino acid residue. MS analyses commonly involve proteolytic digestion of a mixture of proteins into numerous peptides. The peptides can be directly ionized by matrix-assisted laser desorption ionization (MALDI) or electrospray ionization (ESI), or ionized by either of these methods following on-line (ESI) or oV-line (MALDI or ESI) chromatographic separation by reverse-phase liquid chromatography (LC). The presence of modiWcations can be inferred from diVerences in the mass-to-charge ratios (m/z) observed for modiWed and unmodiWed amino acid residues. Peptides containing a single site of modiWcation can be analyzed by MS using parent ions, *
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which are ions carrying an extra proton (MH+). However, if more than one site of modiWcation is present within a peptide, tandem mass spectrometry (MS/MS) is required. In an MS/MS experiment, the peptide backbone is fragmented in a process called collision-induced dissociation (CID). Bond breakage occurs predominantly at amide bonds (usually at one site per molecule) creating a mixed population of N-terminal and C-terminal ions, which are referred to as b and y ions, respectively. (A useful tutorial can be found in [5].) For mass spectrometry to be used quantitatively, it is important that proteolysis, ionization, and fragmentation occur equivalently for both modiWed and unmodiWed peptides. One way to ensure equivalency is to use isotopic labeling which creates a chemically identical population of molecules that can be distinguished by mass. The method described in this article combines CID MS/MS and isotopic labeling to directly measure endogenous levels of acetylation at individual lysine residues. This method was initially used to compare the level of acetylation between wild-type histone H4 and H4 mutants that increased telomeric silencing [6]. The data presented below come from the analysis of an H4
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mutant that has a G7 ! I mutation (Fig. 1A). This mutant (called H4_G7I) was isolated from yeast cells using a procedure established by Waterborg [7]. Since histone proteins isolated from cells are variably acetylated, it was necessary to create a chemically identical population of histones that could be used in a quantitative analysis. This was accomplished by acetylating, in vitro, all unmodiWed lysine residues with deuterated acetic anhydride. Since the mass diVerence between a protiated acetyl group (42 Da) and a deuterated acetyl group (45 Da) is 3 Da, it was possible to distinguish by mass spectrometry between lysine residues that were acetylated in vivo and those that were acetylated in vitro. A schematic showing the acetylation procedure for histone H4 is presented in Fig. 1B. Following chemical acetylation, H4_G7I was digested with the protease trypsin. Normally trypsin cleaves the peptide backbone following lysine and arginine residues. However, the presence of acetyl groups on lysine residues prevents tryptic cleavage at these sites. The four lysine residues within the amino terminal tail of histone H4 were thus encompassed in a single proteolytic fragment containing residues 4–17, abbreviated as H4_G7I(4–17). One of the challenges in quantifying acetylation within the amino terminal tails of histones is the abundance of lysine residues in close proximity to one another. For this reason, CID-MS/MS was required to fragment the parent ion into smaller b and y ions containing one, two or three acetylated sites. The 3 Da diVerences in mass observed for both parent and fragment ions allowed us to determine the levels of acetylation at individual lysine residues. To understand how data from MS and MS/MS analyses provided information about the acetylation levels of ions containing more than one acetylated lysine residue, consider that an ion being analyzed contains n modiWed residues. Each of the residues can be in one of two states: H represents the presence of a protiated acetyl and D represents the presence of a deuterated acetyl. There are only two possible states for an ion with only one modiWed residue (H or D) and therefore only two clusters of m/z peaks in the mass spectrum of such ions. (Each cluster has multiple peaks due to the natural abundance of heavy isotopes such as 13 C, 15N, and 18O. The term ‘monoisotopic’ is used below to describe the Wrst peak in the cluster.) To determine the level of in vivo acetylation at this particular site, one need only determine the ratio of H to the total amount of H and D, that is H/(H + D). This simple analysis is not possible for an ion containing more than one modiWed residue. For an ion with two modiWed residues, there are four possible states: HH, HD, DH, and DD; however, HD and DH will have the same mass so they are indistinguishable by MS. Thus, in the mass spectrum of such an ion, there are three monoisotopic m/z peaks. Extending this further, for an ion with three modiWed residues, there are eight possible states (HHH, HHD, HDH,
DHH, DDH, DHD, HDD, and DDD) of which two groups of three have the same mass (HHD, HDH, and DHH and DDH, DHD, and HDD), resulting in four monoisotopic m/z peaks. Similarly, for an ion with four modiWed residues, there are 12 possible states, resulting in Wve m/z peaks. Thus, for an ion with n modiWed residues, there will be n + 1 monoisotopic m/z peaks in the mass spectrum of that ion. Mathematical expressions, showing how the level of acetylation in parent and fragment ions containing multiple modiWed residues can be determined from other ions with fewer modiWed residues, are described in detail in Smith et al. [8]. In this previous work, we showed that the fraction of in vivo acetylation at a particular site can be determined by summing up all of the in vivo acetylations (normalized) within a given CID fragment and subtracting from this the fraction of in vivo acetylation at all sites other than the one of interest. In essence, the total number of in vivo acetylations within a given fragment was determined by “weighting” each of the peaks according to the number of protium acetylations represented by that peak. This sum was then normalized by dividing by the sum of the intensities for all species (HHHH through DDDD) and subtracting from the total the fraction of acetylation at each site for which the fraction of acetylation had been determined using other types of CID fragment ions. Since an in-depth treatment of theory behind the quantitative aspects of this method has been published elsewhere [8], the goal of this article is to serve as a more practical guide for carrying out this method.
2. Description of method 2.1. List of materials 1. Proteins puriWed by reversed-phase HPLC (see description below). 2. Deuterated acetic acid (Acros, #166210050) and deuterated acetic anhydride (Acros, #174670050). 3. TPCK-treated trypsin (Worthington, #LS003740) and ammonium bicarbonate (Sigma, #A6141). 4. LC electrospray ionization ion trap mass spectrometer. (We have used ThermoFinnigan LCQ instruments at Fred Hutchinson Cancer Research Center in Seattle, WA; the Nebraska Center for Mass spectrometry in Lincoln, NE; and the laboratory of Dr. Larry David at Oregon Health and Science University using both electrospray and MALDI (MassTech, Columbia, MD) sources.) 5. ThermoFinnigan Xcalibur and Microsoft Excel software. 6. HPLC grade acetonitrile (Fisher, #A998-4) with 0.1% triXuoroacetic acid (Sigma–Aldrich, #T62200) or 0.2% acetic acid (Fisher, A38-212).
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Fig. 1. Flowchart describing the experimental procedure used to quantify site speciWc levels of histone H4 acetylation. (A) Amino terminal tail region of the histone H4_G7I mutant containing four lysine residues, located at positions 5, 8, 12, and 16. In most species the N-terminus is blocked by acetylation. The arrows above the sequence indicate sites of tryptic digestion (following complete acetylation of histone H4, see below) that generate the H4_G7I(4–17) peptide. (B) Schematized population of histone H4 molecules; open circles indicate the presence of an in vivo acetylation. Following HPLC puriWcation, the histone H4 proteins are treated with deuterated acetic anhydride to acetylate all lysine residues not acetylated endogenously; the closed circles indicate the presence of a deuterated acetyl moiety. Treatment with trypsin results in cleavage following arginine (R) 3 and R17 (see (A)), producing a chemically identical population of histone H4_G7I peptides, containing residues 4–17, for which the lysine residues acetylated in vivo are tagged with a protiated acetyl group and those that were acetylated in vitro are tagged with a deuterated acetyl group. Since there is a 3 Da mass diVerence between protiated and deuterated acetyl groups (42 Da versus 45 Da, respectively), the endogenous level of acetylation can be determined using mass spectrometry.
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7. Speed vacuum microfuge and temperature-controlled bath. 2.2. Obtaining puriWed histones The yeast histone puriWcation procedure established by Waterborg [7] has repeatedly produced excellent protein samples for MS analysis. A description of Waterborg’s puriWcation of histones from crude yeast nuclei can be found at http://www.fhcrc.org/labs/gottschling/ yeast/. Our only deviation from this procedure involved adding the deacetylase inhibitor zinc sulfate (at a Wnal concentration of 2 mM) to the nuclear isolation buVer. After isolating the Xow-through from centricon devices, histone containing fractions were combined in microfuge tubes and dried down to »100 L using a speed vacuum microfuge. These proteins were then injected onto a Zorbax 150 £ 4.6 mm C-18 column (Alltech, catalogue #883995-902) and eluted using a gradient that increased from 2 to 40% acetonitrile (ACN)/0.1% triXuoroacetic acid (TFA) over 10 min and then increased from 40 to 60% ACN/0.1% TFA over a 40-min period. The Xow rate was maintained at 1 mL/min throughout the separation. Under these conditions, histone H4 co-eluted with H2A at »43% ACN/0.1% TFA and histone H3 eluted »50% ACN/0.1% TFA. To be certain about the identity of these proteins when performing the puriWcation for the Wrst time, SDS–PAGE and Western blot analysis using antibodies to histones should be used (these antibodies can be purchased from Upstate). HPLC puriWed histone samples were then dried in microfuge tubes using a vacuum concentrator and stored at either 4 or ¡20 °C; the protein pellets were either clear or white. 2.3. In vitro acetylation PuriWed and dried histone samples were resuspended in 50 L deuterated acetic acid and 5 L deuterated acetic anhydride and left at room temperature for 6 h. Although this reaction is carried out under acidic conditions, it proceeds to completion because of the large change in free energy resulting from the conversion of an anhydride and amine to a carboxylic acid and amide. The acetic anhydride also acetylates amino acids containing hydroxyl residues; however, this occurs to a lesser extent than with amines. Nonetheless, one should be aware of any serine, threonine, and tyrosine residues in peptides being analyzed by this method. If this is the case, the esteriWed residues can be hydrolyzed under mild alkaline conditions, leaving only the acetylated lysines [9]. Prior to quantiWcation, the degree of acetylation should be ascertained. This can be accomplished by determining the mass of the whole (undigested) protein following the acetylation step. When a mutant form of histone H4 was acetylated and analyzed by mass spec-
trometry, we found six peaks diVering from one another by 42–45 Da; the largest peak occurred at the mass of the protein if all 11 lysine residues of histone H4 were acetylated (data not shown). The peaks representing masses higher than the fully acetylated protein were likely due to acetylation of serine and threonine residues (see above). Since there were no serine, threonine, and tyrosine residues within the amino terminal tail of H4_G7I(4–17), we did not carry out an alkaline hydrolysis step. 2.4. Trypsin digestion Following acetylation, samples were completely dried in a speed vacuum microfuge to evaporate the acetic acid and acetic anhydride, resuspended in 20 mM ammonium bicarbonate with 100 ng trypsin (in 50 L Wnal volume), and incubated at 37 °C overnight. Prior to MS analysis, the ammonium bicarbonate buVer was evaporated using the speed vacuum microfuge. Our MS analyses indicated a signiWcant level of acetylation at the newly formed N-terminus of the H4-G7I(4–17) typtic peptide (see Fig. 1A). This was likely the result of reaction of the amino group of Gly4 with residual acetic anhydride. To eliminate this problem, the acetic anhydride and acetic acid should be neutralized by the addition of mild base prior to evaporation and digestion with trypsin. 2.5. Mass spectrometry There are a number of ways that MS can be used to determine the masses of peptides containing protiated and deuterated acetyl groups. We have investigated both ESI and MALDI of our samples using the LCQ ion trap instrument. While both methods provided excellent MS data, we found that the singly charged ions produced by MALDI did not generate suYcient quality MS/MS spectra after fragmentation in the ion trap. What follows below is a description of the conditions successfully used to analyze the H4_G7I(4–17) peptide with on-line LC followed by ESI and ion trap CID MS/MS. Acetylated and digested histones (H2A and H4) from »500 mL of yeast culture were dissolved in »20 L of 5% formic acid; 2 L of this sample was injected, using an autosampler, onto an Agilent 1100 Series capillary LC system on-line with the ThermoFinnigan LCQ Classic mass spectrometer. In a mobile phase A consisting of 0.2% acetic acid, the sample was loaded onto a 2 cm £ 180 m trap cartridge containing 5 m particle size Zorbax SB-C18 resin (Agilent Technologies) at a Xow rate of 5 L/min. After 10 min of washing, the Xow rate was decreased to 1.5 L/min and the peptides were transferred (on-line) to a 10 cm £ 180 m column containing the same packing material as the trap cartridge. The fused silica of the column lead directly into the Wxed
C.M. Smith / Methods 36 (2005) 395–403
spray tip of the standard ESI source modiWed with a 34 G metal needle (ThermoFinnigan, Cat. No 9714420040). Both trap and column were slurry packed in a 360 m outer diameter fused silica capillary tubing terminated with a microWlter assembly (Upchurch ScientiWc, #M-520). Peptides were eluted with a mobile phase B containing 75% ACN and 0.2% acetic acid. The gradient increased from 2 to 10% B over 10 s and then gradually increased from 10% B to 35% B over 50 min. Under these conditions, the variable isotopic content of the H4 peptides only slightly aVected their elution times. The mass spectrometer began acquiring scans from m/ z 400–2000 (in proWle mode) 10 min after the gradient was started. Under the data-dependent settings, the mass spectrometer was conWgured to acquire MS/MS scans (also in proWle mode) of parent ions with m/z 750.9, which corresponds to the average m/z of the various protiated and deuterated forms of the doubly charged ion of the H4_G7I(4–17) peptide. To activate this feature, the Nth most intense ion from the list button, accessed from the scan event tab, must be activated and a value of 1 entered. The collision energy of CID fragmentation was set at 40% and an isolation mass window of 8 Da was used to isolate all of the isotopic variants—from peptides containing four protiated acetyls to peptides containing four deuterated acetyls. We found that under our condi-
399
tions, a single scan did not provide high enough signal to noise for quantiWcation measurements. However, this problem could be alleviated by averaging several scans. Thus, it is important that this type of mass spectrometer be programmed to perform MS/MS on only the peptide(s) of interest so that the maximum number of scans can be acquired as the peptide elutes from the column. This is accomplished by disabling the dynamic exclusion function of the software and assuring that the minimum MS signal for initiating an MS/MS scan is set suYciently low. It should be noted that oV-line separation followed by MALDI CID recording of the CID spectra might also provide improved signal-to-noise ratios since scans could be acquired on the entire sample. In this case, there would be no chromatographic peak width limitation. 2.6. Data analysis The Xcalibur software that comes standard with the ThermoFinnigan LCQ instrument was used to measure ion currents of parent ions (from MS spectra) and fragment ions (from MS/MS spectra), which in turn allowed us to determine the level of acetylation at various lysine residues. MS data Wles were displayed as two stacked windows containing a plot of the total ion current as a function of time (the chromatogram) on top of a plot of
Fig. 2. Chromatograms of histone H4 and H2A peptides eluting from the on-line LC column. (A) The relative abundance of histone H4 and H2A tryptic peptides, measured by ion current in the ion trap of the mass spectrometer, is plotted as a function of elution time from the on-line column. (B) Chromatogram showing the elution times of peptides within 1 Da of m/z 750.9. Viewing the mass spectrum of the peak at »20 min shows that this peak contains the H4_G7I(4–17) peptide (see Fig. 3).
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the ion current as a function of m/z (the spectrum). Fig. 2A shows the full chromatogram for the histone H2A and H4 mixture. Under the ‘ranges’ option in the chromatogram window, a ‘full ms’ scan Wlter and a ‘mass range’ at 750.9 were selected, showing the time at which peptides of this m/z eluted from the on-line column (Fig. 2B). With the spectrum window active, the mouse cursor was dragged across individual chromatogram peaks causing the averaged MS spectra of these
parent ions to be displayed along with other ions that eluted from the column at this time. The H4_G7I(4–17) peptide eluted »20 min after the MS analysis began; singly and doubly charged H4_G7I(4–17) peptides, in their Wve variably acetylated forms, were seen at m/z »1504 and m/z »752.5, respectively (Fig. 3A). The overall number of deuterated and protiated acetyls were observed by dragging the cursor across the singly charged parent ion (see expanded view in Fig. 3A). To view the averaged MS/
Fig. 3. LC-ESI spectra of isotopically labeled histone H4_G7I(4–17) tryptic peptide. (A) Mass spectrum showing the m/z and relative abundance of all species eluting with the histone H4_G7I(4–17). The most abundant m/z of singly (MH+) and doubly (MH2+) charged H4_G7I(4–17) ions are 1503.9 and 752.5 Da, respectively. Expanded above the spectrum are the clusters of peaks that correspond to the Wve possible protiated and deuterated forms of the fully acetylated isotopically labeled H4(4–17); 0D, 1D, 2D, etc. mark the peaks representing peptides with zero, one, two, etc. deuterated acetyl groups. (B) Tandem mass spectrum (MS/MS) showing the relative abundance of fragment ions of the H4_G7I(4–17). Peaks are labeled according to ion number for the b and y ions described in (C). In some cases, a loss of water caused the mass of the fragment ions to be 18 Da lower than expected (see b7 and b9). (C) Diagram of fully acetylated histone H4_G7I(4–17), showing the masses of b and y ions, indicated above and below the sequence, respectively. The masses represent fragments containing only protiated acetyl groups; each deuterated acetyl group increases the mass by 3 Da.
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MS scans of the 750.9 parent ion, the ‘ms/ms’ Wlter was selected under ‘ranges’ in the spectrum window (Fig. 3B). Close-up views of the peaks in the MS/MS spectrum were obtained by dragging the cursor across speciWc peaks (Fig. 4). This allowed the 3 Da mass diVerences of the various H4_G7I(4–17) fragments to be observed. As discussed in Section 1, ions containing n acetylated lysine residues produce n + 1 m/z peaks. Because of the natural abundance of heavy isotopes, each monoisotopic peak is followed by smaller isotope peaks, each at one a.m.u. higher m/z. The presence of both in vivo and in vitro acetylations within each fragment causes the monoisotopic peaks to diVer from one another in 3 Da increments. To measure the abundance of the various protiated and deuterated forms of a particular b, y or parent ion, ion currents (or intensities) of peaks were measured by dragging the cursor across each cluster of peaks (see Fig. 4A), with the chromatogram window active, and recording in an Excel Wle (see below) the normalized number displayed in the upper right corner of the chromatogram window. As mentioned earlier, under our conditions, the multiple MS/MS scans of the doubly charged peptide were needed to obtain the highest quality peak pattern. Fig. 4B demonstrates how averaging individual scans produced smooth peaks whose ion currents can be readily determined. Not every MS/MS fragment of the parent ion was suitable for quantiWcation, even after averaging; therefore, it was necessary to assess each cluster of peaks before carrying out the quantitation. However, obtaining a suYcient number of quality peaks was not a problem since there were multi-
401
ple b and y ions that could be used to analyze each lysine residue [6,8]. Ion currents of the peaks corresponding to the variably protiated and deuterated forms of b, y, and MH+ ions were entered into columns of an Excel spreadsheet (Table 1). The fraction of each of the variably protiated and deuterated species was determined by dividing the intensity of each species by the sum of the intensities of all the species for a particular ion. For example, for an ion with two lysine residues the fraction of ions with two protiated acetyls is HH/(HH + HD/DH + DD). A formula that executed this calculation was entered into the equation box (G) titled ‘0D’ since the HH ion contains no deuterated acetyls groups. To determine the fractional contribution various acetylated and deuterated species made to the total number of ions of a particular m/z, similar formulas were entered into boxes H, I, J, and K, which were titled ‘1D,’ ‘2D,’ ‘3D,’ and ‘4D.’ Next, the fraction of in vivo acetylation (fac) was determined. For fragments containing one lysine residue, fac is the same value as in the 0D column. For fragments containing more than one lysine residue, fac was determined by multiplying the values in the 0D through 3D columns by the number of protiated acetyls present in the ions they represent and subtracting from this sum the average fac for the other lysines within the fragment ion. Based on the example grid boxes in Table 1, for an ion consisting of two lysine residues the formula was ‘(2 ¤ G + 1 ¤ H) ¡ M2’; for an ion consisting of three lysine residues, the formula was ‘(3 ¤ G + 2 ¤ H + 1 ¤ I) ¡ (M2 + M3)’; and for an ion consisting of three lysine residues, the formula was
Fig. 4. Expanded mass spectra of MS/MS ions of H4_G7I(4–17). (A) Ion intensities were determined from selected ion plots for each isotope cluster (a monoisotopic peak and the following two isotope peaks). Masses of fragmentation ions are indicated. See Fig. 3C for the peptide sequence corresponding to each m/z. The ion current was measured by dragging the cursor across each clusters of three peaks (arrows) and then recording the normalized (NL) values from the chromatogram window (the numbers above each arrow) in Excel spreadsheet boxes (Table 1). (B) The top spectrum is the average of eight individual scans (below); this demonstrates the importance of averaging spectra to maximize peak quality.
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Table 1 Example Excel spreadsheet for determining the fraction of acetylation at lysines 5, 8, 12, and 16 of histone H4_G7I Aa 1y ionb 2y5(530) 3y7(757) 4y10(1097) 5MH+(1494) a b c d e
B
C
D
E
F
G
H
I
J
K
L
M
ICc 1.01E+06 2.08E+05 1.02E+05 2.6E+05
IC 3.03E+05 7.84E+05 5.00E+05 5.4E+05
IC
IC
IC
1D
2D
3D
4D
1.35E+05 5.75E+05 2.0E+06
1.11E+05 2.5E+06
0Dd 0.77 0.18 0.08 0.04
0.70 0.39 0.09
0.12 0.45 0.32
0.09 0.41
0.14
Lys residue 16 12 8 5
fAce 0.77 0.30 0.40 0.02
8.8E+05
Grid box names from Excel spreadsheet are correlated with description in text. y ions, m/z of C-terminal fragments ions. IC, ion current measurements for a particular peak, beginning with the lowest m/z of the set of peaks. 0D, 1D, 2D, 3D, and 4D, the fraction of ions containing zero, one, two, three or four deuterated acetyl groups, respectively. fAc, fraction of acetylation at individual lysine residues.
‘(4 ¤ G + 3 ¤ H + 2 ¤ I + 1 ¤ J) ¡ (M2 + M3 + M4),’ where the M column represents the fac of already determined lysine residues within a particular fragment. Since multiple ions were available for analysis, fac values were averaged and multiplied by 100 to give the average percent acetylation at each lysine residue. An example spreadsheet showing all of the data we acquired for the acetylation analysis of another histone H4 mutant (A15T) is available at http://chem-ncms.unl.edu/SupFile.html.
information presented here is intended to serve in conjunction with Smith et al. [8], which contains a detailed description of the theory behind the quantitation strategy for ions containing more than one site of modiWcation. While this method has focused on acetylation of histones, it could also serve as a guide for the quantiWcation of other modiWcations that are located near one another in the primary structure of a protein.
Acknowledgments 3. Concluding remarks Within the past few years, the number of publications describing the type and location of modiWcations in histones has increased dramatically. To catalogue these numerous modiWcations, researchers have used modiWcation speciWc fragmentation patterns [10], construction of histone modiWcation databases that can be searched using specialized computer programs [11], and high resolution mass spectrometers, such as quadrupole time-of-Xight and ESI Fourier-Transform ion cyclotron resonance instruments [12,13]. Quantitative MS analyses have also been used to investigate changes in the levels of histone H3 methylation and acetylation in mouse embryonic stem (ES) cells [14], yeast [15], and Drosophila Kc cells [16]. The ES cell studies involved the use of chemically synthesized and modiWed peptide standards to account for variability in ionization and fragmentation between samples [14]. The yeast and Drosophila studies by-passed the need for chemical equivalency by comparing the parent masses of modiWed peptides isolated from cells with diVerent genetic backgrounds [15,16]. The method described in this article focuses on the direct and accurate measurement of acetylation at individual lysine residues that are in close proximity to one another in a tryptic peptide, and it involves the use of a ThermoFinnigan LCQ mass spectrometer, which is one of the least costly and most commonly found mass spectrometers in laboratories today. This proteomic approach is diVerent from other quantitative MS approaches of histone modiWcation in that it combines isotopic labeling using deuterated acetic anhydride and CID MS/MS. The
A special thanks to Larry David and Zhongli Zhang for running the G7I mutant sample. Support for the analysis of samples at OHSU was provided by National Eye Institute Core Grant EY10572. Additional thanks go to Larry David and David and Jean Smith for helpful conversations. And my appreciation goes to the Gottschling lab for helping me gather information for this article.
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