Methods 26 (2002) 260–269

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Isolation and mass spectrometry of transcription factor complexes

G. Sebastiaan Winkler,a,1 Lynne Lacomis,b,1 John Philip,b Hediye Erdjument-Bromage,b
Jesper Q. Svejstrup,a,* and Paul Tempstb,*
a
Cancer Research UK London Research Institute, Clare Hall Laboratories, Blanche Lane, South Mimms, EN6 3LD, Hertfordshire, UK
b
Molecular Biology Program, Memorial Sloan–Kettering Cancer Center, 1275 York Avenue, New York, NY 10021, USA
Accepted 1 February 2002

Abstract

Protocols are described that enable the isolation of novel proteins associated with a known protein and the subsequent iden-
tification of these proteins by mass spectrometry. We review the basics of nanosample handling and of two complementary ap-
proaches to mass analysis, and provide protocols for the entire process. The protein isolation procedure is rapid and based on two
high-affinity chromatography steps. The method does not require previous knowledge of complex composition or activity and
permits subsequent biochemical characterization of the isolated factor. As an example, we provide the procedures used to isolate
and analyze yeast Elongator, a histone acetyltransferase complex important for transcript elongation, which led to the identification
of three novel subunits. Ó 2002 Elsevier Science (USA). All rights reserved.

1. Introduction can be expected that factors identified by genetic means

Proteomics is the newest chapter in biological systems
analysis. A more focused embodiment, termed targeted
proteomics, calls for the examination of subsets of the
proteome, e.g., those proteins that either are specifically
modified, or bind to a particular DNA sequence, or exist
as members of higher-order complexes, or any combi-
nation thereof. We have sought to apply this concept to
the study of gene expression.
The genome of eukaryotic organisms contains a
plethora of factors important for the regulation of
transcription. Genetic and biochemical analysis of gene
transcription have identified numerous regulatory pro-
teins, including chromatin-modifying enzymes. In ad-
dition, homology searching in the recently fully
sequenced genomes reveals the presence of many addi-
tional, yet uncharacterized factors that are likely also
important for transcriptional regulation. Because high-
molecular-weight multisubunit complexes have fre-
quently been identified by biochemical approaches, it
* magglutinin (HA) immunoaffinity procedure that allows
Corresponding authors.
E-mail addresses: j.svejstrup@icrf.icnet.uk (J.Q.
p-tempst@mskcc.org (P. Tempst).
1
Authors contributed equally to this review.
or by sequence homology are also functional in the
context of multiprotein assemblies. Here we describe a
generic method that can be used to purify proteins and
their associating partners and their subsequent identifi-
cation by mass spectrometry. The complete procedure
does not require previous knowledge of complex com-
position or function, and is fast, reliable, and suitable
for proteome analysis.
As an example, we provide the protocol used to iso-
late yeast Elongator, a histone acetyltransferase complex
important for transcript elongation, which led to the
identification of three novel subunits by mass spect-
rometry.
2. Methods
2.1. Affinity purification of protein complexes
The procedure presented herein is based on the high
affinity of histidine stretches to Ni-agarose and a he-
Svejstrup),
elution by competition with excess peptide. Both tags
have been used individually in both yeast and mam-
malian systems, facilitating the isolation of multisubunit

1046-2023/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved.
PII: S 1 0 4 6 - 2 0 2 3 ( 0 2 ) 0 0 0 3 0 - 0
 G. Sebastiaan Winkler et al. / Methods 26 (2002) 260–269 261

complexes (see, for example, [1–4]). However, the com-
bination of both tags and the employment of competitor
peptide that allows relatively high yields from the anti-
HA immunoaffinity step improve the use of these tags
for the purification of rare complexes [5,6]. The protocol
is rapid and permits the biochemical characterization of
the isolated factor.
2.1.1. Expression of tagged Elp1 in yeast
We constructed a tagged version of the yeast ELP1
gene, encoding the largest subunit of Elongator. The
tagged gene was expressed at normal levels by replacing
the endogenous chromosomal copy with the tagged se-
quence by homologous recombination in yeast. This
approach makes it possible to avoid the creation of
nonphysiological complexes with altered stoichiometry
as might be caused by protein overexpression. Further-
more, careful phenotypic analysis was carried out to
ensure that the sequence encoding the affinity tag did
not interfere with gene/protein function. To facilitate
carboxyl-terminal genomic tagging, we have developed a
generic vector, pSE-304-HisHA (Fig. 1), in which mul-
tiple unique restriction enzyme sites precede the tag and
a transcription termination signal [7].
2.1.2. Preparation of immunoaffinity reagents
The anti-HA immunoaffinity procedure uses the
mouse 12CA5 monoclonal antibody. These antibodies
can be obtained commercially (Roche), and the 12CA5
hybridoma is also widely distributed among research
laboratories. We used crude (concentrated) cell culture
supernatant or ascites fluid directly to prepare the im-
munoaffinity resin.
We routinely prepared anti-HA immunoaffinity re-
sin by binding 1–3 mg 12CA5 antibody per milliliter
of protein A–Sepharose resin (Amersham–Pharmacia
Biotech) equilibrated in phosphate-buffered saline
(PBS) for 30 min at room temperature with continu-
ous mixing. As the elution conditions do not interfere
with the antibody–protein A binding, the antibodies
were not covalently crosslinked to the resin. This al-
lows the recovery of protein A–agarose resin by re-
moving antibody–peptide complexes with 0.1 M
glycine (pH 2.5).
2.1.3. Purification of double-tagged Elp1 protein from
yeast cells
Protease-deficient yeast cells BJ2168 (MATa, prc1-
407, prb1-1122, pep4-3, leu2, trp1, ura352, gal2) [8] ex-
pressing a HisHA-tagged version of the ELP1 gene
were grown to late-log phase and lysed with glass beads,
and whole-cell extracts were prepared as described [7,9].
We found that the yield and purity of the isolated
material were greatly improved by starting with chro-
matography on a conventional resin, such as Bio-Rex
70 (Bio-Rad). This resin was chosen because most
DNA-related factors bind to it, but other resins, such as
heparin–Sepharose (Pharmacia–Amersham), could also
be used. The Bio-Rex fraction containing HisHA-
tagged Elp1 was then incubated with anti-HA immun-
oaffinity resin (Fig. 2A). In some instances, soluble
whole-cell extract was incubated directly with the im-
munoaffinity resin.
1. Incubate portion of the Bio-Rex 70 fraction contain-
ing the majority of Elp1 protein (50 mL, 300 mg total
protein) in buffer A (40 mM Hepes–KOH, pH 7.6, 1

Fig. 1. Plasmid pSE.HisHA-304. Indicated are the bacterial origin of replication and the ampicillin resistance gene, the TRP1 marker, and the
promoter-less HisHA tag followed by a transcription termination signal derived from the ADH1 gene. Also shown are the nucleotide and amino acid
sequences of the polylinker fused to the decahistidine stretch and HA epitope tag.
262 G. Sebastiaan Winkler et al. / Methods 26 (2002) 260–269

Fig. 2. Affinity purification of the Elongator histone acetyltransferase complex. (A) Schematic diagram of the HisHA-tagged Elp1 protein and the
purification strategy. (B) SDS–PAGE analysis of protein fractions from the anti-HA immunoaffinity column. Top: Immunoblot. The HisHA-tagged
Elp1 protein was detected using rat monoclonal antibody 3F10 recognizing the HA epitope (Roche). Bottom: Protein silver staining. Indicated on the
left are positions of the size markers. (C) Analysis of protein fractions from the Ni-NTA affinity column by protein silver staining. Indicated on the
left are positions of the size markers.

mM EDTA, 1 mM dithiothreitol, 20% (v/v) glycerol)
containing 600 mM potassium acetate several hours
to overnight at 4 °C with 0.8 mL anti-HA immuno-
affinity resin with continuous, gentle mixing.
2. Collect the resin by gravity flow in a disposable poly-
propylene column holder (Econo-Pac, 1:5
12 cm,
Bio-Rad). Reapply the flow-through twice onto the
resin.
3. Wash the immunoaffinity resin twice with 10 mL
buffer A containing 600 mM potassium acetate.
4. Subsequently equilibrate the column in buffer E (40
mM Hepes–KOH, 1 mM 2-mercaptoethanol, 20%
(v/v) glycerol) containing 300 mM potassium ace-
tate.
5. Finally, elute bound protein with excess peptide.
Gently mix the resin with 1 mL prewarmed buffer
 G. Sebastiaan Winkler et al. / Methods 26 (2002) 260–269
E containing 300 mM potassium acetate and 1 mg/
mL HA peptide (KKKRILKMYPYDVPDYARIL)
and submerge the column holder in a water bath at
30 °C for 15 min. Occasionally, resin and buffer were
mixed by gentle tapping. Collect the eluate by grav-
ity flow and immediately place on ice. Repeat this
step twice.
Protein fractions were analyzed by SDS–PAGE and
stained with silver nitrate (Fig. 2B). In addition to
Elp1 and two known Elongator subunits, Elp2 and
Elp3, three additional associating proteins with ap-
parent molecular masses of 50, 35, and 30 kDa were
identified. These proteins do not bind to anti-HA im-
muno-affinity resin in the absence of HisHA-tagged
Elp1. Thus, virtually homogeneous Elongator complex
was obtained in a rapid two-step procedure. However,
a second high-affinity step using the decahistidine
stretch was used to verify that these proteins are indeed
associating with Elp1. This second affinity step can
also be used to concentrate the protein fractions and is
required when crude extract is incubated directly with
anti-HA immunoaffinity resin, or when the target
complex is present at relatively low levels in the ex-
tract. Moreover, the Ni-agarose efficiently removes the
excess HA-peptide used for elution from the anti-HA
resin, which might interfere with mass spectrometric
analysis.
1. Pool the eluted fractions from the anti-HA immuno-
affinity column and incubate several hours to over-
night with 0.4 mL Ni-NTA agarose (Qiagen) at
4 °C with continuous mixing.
2. Collect the Ni-agarose resin by gravity flow in a dis-
posable polypropylene column holder (Polyprep,
0:8
4 cm, Bio-Rad). Reapply the flow-through
twice onto the resin.
3. Wash the Ni-agarose resin once with 1 mL buffer E
containing 300 mM potassium acetate, and twice
with 1 mL buffer E containing 300 mM potassium
acetate and 10 mM imidazole.
4. Finally, elute bound protein with three washes of 0.4
ml buffer E containing 300 mM potassium acetate
and 300 mM imidazole.
All procedures were carried out at 4 °C.
Protein fractions were analyzed by SDS–PAGE and
stained with silver nitrate (Fig. 2C). The associating
proteins with apparent molecular masses of 50, 35, and
30 kDa coeluted from the Ni-NTA agarose column.
These proteins were prepared for identification by mass
spectrometry and shown to be bona fide subunits of
Elongator [7].
2.2. Identification of proteins by mass spectrometry
Proteins isolated in the manner described in the pre-
vious section usually yield a small amount of protein,
often less than 1 pmol per band. Whereas chemical
263
microsequencing has long been the technique of choice
for protein identification [10], femtomole-level analysis
can only be done using a mass spectrometric approach.
‘‘Mass fingerprinting’’ uses a small set of proteolytic
fragment masses—five or six, but accurate to within 30–
50 ppm—to establish identity [11,12]. Identification is
most easily done by matrix-assisted laser desorption/
ionization (MALDI) reflectron time-of-flight (reTOF)
mass spectrometry (MS). Such a data set does not,
however, allow querying an expressed sequence tag
(EST) database, nor is it usually enough to cope with
mixed or contaminated proteins. In such instances, a
multimode MS approach, particularly including elec-
trospray ionization (ESI) tandem MS, is preferred [13].
Tandem MS enables protein identification on the basis
of fragmentation data from a single, derivative peptide
[14,15].
MS-based protein identification is multistep and all
steps are critical to the successful outcome of the anal-
ysis. In general, mass spectrometers are not very tolerant
of particulates, salts, detergents, buffer components, and
any or all ionizable molecules other than peptides (this
in case of peptide analysis, of course). Furthermore, it is
a concentration-sensitive technique, requiring the sam-
ple to be presented for analysis in the smallest possible
volume. As a rule, sample handling and adsorptive
losses to tubes and pipet tips should be kept to a
minimum, chemical modifications avoided, and no ex-
traneous proteins introduced. Using the purified Elon-
gator complex as an example, we consider these steps in
turn.
2.2.1. In-gel tryptic digestion
The purified Elongator-associated proteins were re-
solved by 10% SDS–PAGE and stained with Coomassie
Blue R–250. Tryptic digests were carried out using the
following protocol (Coomassie-stained gel piece 6 10

3
1 mm).
1. Destain (plus remove SDS) with 50% methanol (v/v),
vortex, place in 37 °C bath for 15 min, spin down,
and remove supernatant. Repeat three times.
2. If still blue, wash with 50% acetonitrile in 0.2 M am-
monium bicarbonate.
Note: All color and SDS must be removed from the
gel pieces or the residual stain will cause peptide losses
during later RP microtip step.
3. Rinse the gel four times with Milli-Q water.
4. Using a designated (and clean) Petri dish, tweezer,
and scalpel, dice the gel to  1-mm3 pieces.
Note: It is important that the gel does not become too
dry during this procedure; otherwise it will be difficult to
gather the pieces since they become hardened and sticky.
On the other hand, the gel should not be too wet because
this will delay drying down and promote losses (see next
step).
5. Speed-Vac the sample to dryness for 15 min.
264 G. Sebastiaan Winkler et al. / Methods 26 (2002) 260–269
6. Once the gel pieces are semidry to bone dry, add
0:2 lg trypsin (Promega; modified trypsin, porcine)
in 5 lL digest solution: 0.02% (w/v) Zwittergent 3–
16 (Calbiochem) in 0.1 M ammonium bicarbonate
(NH4 HCO3 ).
7. Allow sufficient time for the enzyme solution to be
entirely adsorbed by the dry gel.
8. Add sufficient digest solution (Zwittergent included)
to reswell the gel pieces to the original volume and
then completely submerge them. However, the final
volume of supernatant should ideally not exceed
50 lL.
Note: The precise (0.02%) Zwittergent 3–16 concen-
tration and supernatant volume ð650 lLÞ are critical for
the success of the microtip cleanup procedure.
9. Incubate for 2 h at 37 °C.
10. Sonicate (in bath) for 5 min; spin down for 1 min
in Eppendorf centrifuge.
11. Draw up the entire supernatant (<50 lL) and im-
mediately deposit on a 2-lL bed volume RP micro-
tip for cleanup (see text). This will then provide
desalted, concentrated peptide pools for MALDI
re-TOF MS and CF NanoES-MS/MS analysis.
The entire procedure should ideally be done inside a
‘‘clean box,’’ for instance, a PCR AirClean Systems
workstation, and using dedicated handling tools and
plasticware. It is especially important that all dye and
SDS be removed from the gel pieces as we have ob-
served these to adversely affect sample cleanup (mi-
crotip) afterward, resulting in low recoveries. Note
that, after the initial drying and reswelling in trypsin
containing buffer, no further drying steps are included
anywhere in the procedure, all the way to the intro-
duction into the mass spectrometers. We strongly rec-
ommend the inclusion of 0.02% Zwittergent 3–16 (a
zwitterionic detergent) in the digest buffers for its un-
ique ability to prevent adsorptive losses of even low-
femtomole amounts of peptides [16]. Zwittergent is
conveniently, and completely, removed by passage over
a reversed-phase microtip, a step already required for
peptide desalting and concentration. However, care
should be taken not to saturate the RP tip. As a rule, a
2-lL bed volume of Poros R2 beads can easily bind all
Zwittergent from 50 lL of a 0.02% solution (or 1 lL
of 1% Zwit). As such, there are limitations on what
volume of gel pieces can be processed. Combining
several bands from parallel lanes in a gel may result in
volumes exceeding 50–100 mm3 , requiring 100–200 lL
of digest buffer. Aside from the dilution factor and
losses, the total amount of Zwittergent will here-
by exceed microtip capacity, impeding sample pre-
paration.
2.2.2. Modified silver stain and bleach
Gel bands stained with Coomassie R-250, or its
colloidal form (Pierce), are ideal for MS-based protein
identification. The Coomassie stain detection limit of
an average band, <1 cm wide on a 0.5- to 1-mm-thick
gel, is about 25–50 ng. For proteins of molecular
weights under 50 kDa this amount represents about 1
pmol or more; in excess of what is ideally needed for
simple mass spectrometric identification (0.5 pmol).
In these cases, silver staining will be necessary and is
recommended. However, some restrictions apply. The
proteins under study should not be chemically modi-
fied, except perhaps to reduce and alkylate cysteine
residues, which may be helpful in some cases for the
digest to proceed optimally. Inadvertent covalent
modifications will result in changes in peptide molecu-
lar mass, precluding ‘‘peptide fingerprinting’’-type dat-
abase searches (see below). Treating proteins with an
excess of aldehydes, a procedure commonly used in
most commercial silver stains, may result in Schiff base
formation with primary amines such as lysine side
chains, interfering with subsequent tryptic digestion (at
the C terminus of Lys and Arg) and accurate mass
measurement. A modified silver stain has been devel-
oped [17] to minimize such effects. Second, it has been
noted that silver staining, including the modified
version, has adverse effects on overall peptide recov-
ery after digestion. This effect progressively wors-
ens with the duration of exposure (i.e., development
time), as shown by comparative, quantitative analy-
sis (L.L., H.E.B., and P.T., unpublished results). It can
be remedied, to some extent, by bleaching the silver
[18].
1. Fix gel in 40% ethanol/10% acetic acid in water (v/v),
overnight.
2. Wash for 10 min in 50% methanol (v/v).
3. Wash with water for 10 min to remove residual
acid.
4. Sensitize gel by a 1-min incubation in 0.02% (w/v)
sodium thiosulfate (Sigma, Catalog No. S8503).
5. Rinse with two changes of Milli-Q water for 1 min
each.
6. Submerge gel and incubate in 0.1% (w/v) silver ni-
trate (Aldrich Chem. Co., Catalog No. 20,505-2)
for 20 min at 4 °C (e.g., cold room).
7. Discard silver nitrate and rinse gel twice with Milli-Q
water for 1 min each.
8. Develop gel in 0.04% formaldehyde [dilute 108 lL of
commercially available 37% formaldehyde (Sigma,
Catalog No. F-1268) in 100 mL of 2% (w/v) sodium
carbonate (Fisher Sci., Catalog No. S263-3)] under
vigorous shaking.
Note: After the developer turns yellow, it should be
immediately discarded and replaced with fresh solution.
9. Terminate the development by discarding the re-
agent and storing the gel in 1% acetic acid at 4 °C
until further analysis.
Note: Storage in glycerol solution is not recom-
mended, as it interferes with proteolytic digests.
 G. Sebastiaan Winkler et al. / Methods 26 (2002) 260–269 265

Table 1
Mass spectrometric protein identification: Sloan–Kettering System
1. Protein(s) in gel (see text)
2. Tryptic digest (see text)
3. Peptide pools, 3–4 lL volume (see text)
4. MALDI-reTOF MS (take 0.5 lL of step 3)
4.1. Extract mass (m=z) list; designate major, medium, minor peaks
4.2. Subtract background peaks (trypsin autolytic, keratin,. . .): go to step 5.1
5. MALDI List(s) and SEARCH (‘‘Peptide Fingerprinting’’)
5.1. Master list
5.2. Subtracted list(s)
5.2.1. Sub-list 1
5.2.2. . Sub-list 2
..
5.2.n. Sub-list n; until no more unaccounted peaks
5.3. SEARCH
5.3.1. Programs: PeptideSearch (EMBL, Protana); MASCOT (Matrix Sci.)
5.3.2. Parameters: 30–50 ppm accuracy; 1–2 missed cleavage sites
5.3.3. Database(s): NR (NCBI)—integral, or species-separated go to step 6
6. Identification
6.1. Yes (top match clearly separated from rest): go to step 7
6.2. Maybe (top match not much separated from lesser matches, but separated from noise): go to step 8
6.3. No (no top match; some matches can be separated from noise, or not at all):
6.3.1. Perform ‘‘Functional’’ inspection of list by (i) domain expert or (ii) use of PINdb
6.3.2. Candidate proteins? (TFs, coactivators, repressors, other nuclear proteins, etc.): go to step 8
6.3.3. Still no candidates: go to step 9
7. Reinspect MALDI spectra
7.1. All peaks accounted for? Matched peaks: major/medium/minor (see step 4.1)? If Yes: STOP
If NO: go to step 7.2
7.2. Subtract matched peaks from master list (step 5.1); create new Sub-list 1; 2; 3; . . . ; n: go to step 5.2 and repeat search
8. Confirm fingerprint ID by ESI-MS/MS
8.1. Take MALDI master step 5.1 or subtracted (step 5.2): List; convert major m/z from MHþ to MH2þ
and MH3þ ; select
8.2. CF NanoESI-MS/MS (1; 2; 3; . . . ; peptides); take 1.5 lL of step 3
8.3. Confirmed?
8.3.1. Yes: go to step 7
8.3.2. No: go to step 6.3
9. CF NanoESI-MS (and MS/MS) (take 1.5 lL of step 3)
9.1. ESI-MS: find major precursor ions; compare with master list (step 5.1) or subtracted list (step 5.2); select precursor ions for
MS/MS
9.2. MS/MS
9.3. SEARCH (with fragment ions)
9.3.1. Programs: SequenceTag (EMBL; Protana); PepFrag (PROWL, Proteometrics)
9.3.2. Input: m=z (MALDI), y 00 -ions
9.3.3. Databases: NR/species (NCBI); dbEST (NCBI)
9.4. Identification
9.4.1. Yes (single ID; clear y 00 -ion series);
Confirm with 2nd (and 3rd. . . MS/MS): go to step 9.1 pick precursor ion predicted to match putatively identified protein
9.4.1.1. Yes: go to step 7
9.4.1.2. No: go to step 9.1 and start over
9.4.2. No (no signal; noninterpretable; or many ‘‘IDs’’): go to step 9.1 and pick other precursor ion

10. Once bands/spots of interest have been excised from
the gel, prepare destain (‘‘bleach’’) solutions (A and
B) fresh each time, as follows:
A. 30 mM Potassium ferricyanide ½K3 FeðCNÞ6 
(Sigma): Weigh out 0.05 g and dissolve in 5 mL Milli-Q
water.
B. 100 mM Sodium thiosulfate pentahydrate
ðNa2 S2 O3  5H2 OÞ (Sigma): Weigh out 0.0124 g and
dissolve in 5 mL Milli-Q water.
11. Mix equal (500-lL) volumes of solutions A and B,
cover each gel piece with destain solution, and vor-
tex lightly until stain disappears.
266 G. Sebastiaan Winkler et al. / Methods 26 (2002) 260–269
12. Rinse approximately five times with water.
13. Cover each gel piece with 0.2 M NH4 HCO3 in 50%
acetonitrile (v/v).
14. Incubate at 37 °C for 15 min.
15. Rinse approximately five times with water.
16. Cut (dice) gel band and prepare as usual for digest
(see above).
It is recommended that this procedure be followed
closely, as even small deviations may result in later
failure to identify the proteins. As a rule, gels should
only be lightly stained with silver, which will pick up
protein bands in the very low nanogram range. Any-
thing below that may not be enough for analysis any-
way. Proteins of apparent molecular mass over 100 kDa
should be visible either by Coomassie or after very brief
silver development to ensure successful identification.
When not sure, Coomassie staining can be done first
followed by silver staining.
2.2.3. Peptide sample preparation
The digest mixture resulting from in-gel proteolysis
cannot be directly introduced into a mass spectrometer
as it is too dilute and contains many constituents in-
terfering with analysis. Sample cleanup and concen-
tration is now almost universally done using reversed-
phase microtips (i.e., with 1- to 2-lL bed volumes),
either homemade devices [19] or commercially available
Zip-Tips (Millipore). As a result, the peptide mixtures
are then presented for analysis while dissolved in 0.1%
formic acid/30% acetonitrile (in water); the solvent re-
quired for elution from the reversed-phase beads. Or-
ganic solvents promote easy desolvation and formation
of gaseous ions, a prerequisite for mass analysis during
electrospray ionization. Acid promotes solubility; and
the low pH ensures almost universal protonation of all
peptides, enabling operation of any type of mass
spectrometer in the positive ion mode at all times. In
general, we elute peptides from the RP tip in two bat-
ches of 3–4 lL each; the shorter and hydrophilic ones
at 16% acetonitrile and the longer and hydrophobic
ones at 30%. We refer to these successive eluates as the
‘‘16% pool’’ and ‘‘30% pool.’’ As with all other forms
of reversed-phase packings and chromatography, SDS
has a very detrimental effect on analyte separation and
recovery, and should therefore be fully removed prior
to loading of the tip. Further details about the proce-
dure are beyond the scope of this report and can be
found in an earlier publication by some of the authors
[19].
2.2.4. MALDI-TOF mass spectrometry
A homogeneous protein, with archived sequence,
contained within a single gel band in P0:25- to 0.5-
pmol amounts, and prepared for mass spectrometric
analysis as described in the previous sections can be
readily identified by simple peptide mass fingerprinting
as outlined in Table 1 (steps 4–7). For MALDI-TOF
MS, the sample is introduced as a solid crystal, usually
in a 0.5-lL volume or less. This technique is relatively
simple, very accurate (in reflector mode), and very sen-
sitive. For example, the 50-kDa Elongator-complex
protein that eluted from the Ni-NTA column yielded a
distinct series of tryptic peptide ions in the combined
(16% + 30% pools) spectra (Fig. 3). Twelve of the fifteen
selected, major peaks had m=z values that matched, to
within 40-ppm accuracy, the calculated monoisotopic
masses of predicted, singly charged [MHþ ] tryptic pep-
tides of the yeast hypothetical protein YRL101w (the-
oretical molecular mass of 51,156 Da), providing 26%
sequence coverage, with stretches of double or triple
overlap. By contrast, random matches (‘‘noise’’) with all
other yeast proteins of molecular mass 6100 kDa, at
40-ppm accuracy and with at maximum one missed
cleavage site, numbered 63 of 15 (with less than 8%
sequence coverage and no overlaps). The three unac-
counted m=z values could not be matched to any other
yeast proteins and must therefore derive either from
modified peptides or from a nonyeast source. In our
experience, an identification as presented here is to be
considered one of ‘‘high confidence’’ (see step 6.1 in
Table 1). Experimental details of the analysis and dat-
abase search can be found in Fig. 3.
2.2.5. Multimode mass spectrometry
In many instances, initial MALDI-TOF analysis and
peptide fingerprint searching will not yield a clear-cut
result, in that two or more proteins appear as top can-
didates (Table 1, step 6.2); sometimes no real candidates
emerge (Table 1, step 6.3). Several options are available
at this point, as presented in Table 1; all require addi-
tional analysis using tandem mass spectrometry (also
known as MS/MS). This could be either to confirm a
lead (Table 1, step 8) or to make a completely inde-
pendent attempt at identification (step 9). Leads to
identifying a protein may be obtained from inconclusive
‘‘first-pass’’ peptide fingerprint searches, whereby the
return list has been inspected for potential candidates on
the basis of ascribed function (e.g., transcription) or
cellular location (e.g., nucleus).
Most commercially available tandem mass spec-
trometers use electrospray ionization (ESI), which in-
volves introduction of the sample in liquid form and,
ideally, at a very low flow (nanoliters/minute), coupled to
triple-quadrupole and hybrid quadrupole-TOF mass
analyzers. It enables protein identification on the basis of
fragmentation (mass) data derived from a single tryptic
peptide [14,15]. This peptide need not be purified before
analysis as the MS/MS method allows selection of pep-
tides of any particular mass out of complex mixtures.
Subsequent fragmentation results from high-speed col-
lisions with gas molecules, which induce cleavages across
the amide backbone of a peptide and provide ion series
 G. Sebastiaan Winkler et al. / Methods 26 (2002) 260–269 267

Fig. 3. Identification of Elongator ‘‘p50’’ by MALDI-reTOF MS and peptide mass fingerprint search. The gel-bound protein was digested and
peptides were prepared for mass analysis as described in the text. MALDI-reTOF mass spectra of the (A) 16% peptide pool and (B) 30% pool. Note
that the spectra shown here were the result of a duplicate analysis in the absence of calibrants. Major peaks corresponding to m=z (mass-to-charge)
values that could be matched to predicted tryptic peptides from yeast protein YRL101w are labeled with closed circles; those that remained un-
accounted for are indicated with open circles. Experimental: Each peptide pool was analyzed twice by MALDI-reTOF MS, in the presence and
absence of peptide calibrants [19]. Aliquots (0.5 lL) were deposited on the probe surface, mixed with a-cyano-4-hydroxycinnamic acid solution
(MALDI-Quality, Bruker-Daltonics, Billerica, MA) on the plate, and allowed to dry at room temperature; calibrants were diluted from concentrated
stocks and mixed to yield 6.25 fmol of each per 0.2-lL volume of the same solvent prior to mixing with the analytes. Spectra were acquired on a
REFLEX III (Bruker-Franzen, Bremen, Germany) instrument equipped with a 337-nm nitrogen laser, a gridless pulsed-extraction ion source, and a
2-GHz digitizer. The instrument was operated in reflector mode; 25-kV ion acceleration, 26.25-kV reflector, and )1.4-kV multiplier voltages were
used. Ion extraction was done 200 ns after each laser irradiance by pulsing down the source extraction lens to 17.7 kV from its initial 25-kV level to
give appropriate time-lag focus conditions at the detector. Spectra were obtained by averaging multiple signals; laser irradiance and number of
acquisitions (typically 150) were operator adjusted to yield maximal peak deflections, derived from the digitizer as TOF data and displayed in real
time as mass spectra using a SPARC Station 5 (Sun Microsystems; Mountain View, CA). After recalibration with internal standards, monoisotopic
masses were assigned for all prominent peaks (marked with closed or open circles) over background, and a peptide mass list was generated. This list
was taken to search the yeast segment of a protein nonredundant database (NR, National Center for Biotechnology Information, Bethesda, MD)
using the PeptideSearch [11] algorithm. A molecular mass range up to 100 kDa was covered, with a mass accuracy restriction of 40 ppm or better and
a maximum of one missed cleavage site allowed per peptide. The search program can be downloaded over the Internet from the following site: http://
www.narrador.emblheidelberg.de/GroupPages/PageLink/peptidesearchpage.htm/. Or the search can be done directly at the following location: http://
peptsearch.protana.com/FR_PeptideSearchForm.html.

corresponding to the amino acid sequence. The process is
often referred to as ‘‘sequencing,’’ but it rarely ever yields
bona fide sequence (e.g., 10–20 residues) that could be
used for a BLAST search, and then only if ample
quantities are available. Rather, the data are more often
used to confirm a known or surmised structure. Identi-
fications based solely on MS/MS data are certainly
achievable (as will be shown here), but single-peptide
matches, even when statistically sound, must be inter-
preted with caution; two seems a dependable minimum.
A case in point was the analysis of the 30-kDa
Elongator-complex component. MALDI-TOF analysis
provided us with 15 major peptide ions (Fig. 4A) that
were used to search the yeast database, resulting in a list
of seven candidate proteins with matches (P4 of 15)
above background (63 of 15). Three of those had pre-
dicted molecular masses between 29 and 31 kDa,
namely, two ribosomal proteins and one unannotated,
hypothetical protein (i.e., the putative product of an
open reading frame). To find out which of the three
proteins was present, a second aliquot of the peptide
mixture was taken for continuous flow (CF) NanoESI-
MS analysis: first in single stage (Q1) scan mode (Fig.
4B), then for tandem MS of selected precursor ions (Fig.
4C). The MALDI-TOF results were first taken to select
ions that are predicted to correspond to a specific tryptic
peptide from each of the three proteins. MALDI ions
are almost always singly protonated (m ¼ ½M þ Hþ ,
z ¼ 1) and must be converted to the predicted double
(m ¼ ½M þ 2H2þ , z ¼ 2) or triple (m ¼ ½M þ 3H3þ ,
z ¼ 3) positive charge states, expected to result from
electrospray ionization, before locating them on the
ESI-MS (Q1) scan. For instance, for the hypothetical
protein YMR312w, we focused on a MALDI ion of
m=z ¼ 1086:53 (which should correspond to the se-
quence DVTGSLHVCR if the protein was indeed
identified correctly), which should correspond to a pre-
cursor ion of m=z ¼ 543:8 in the Q1 scan (indicated as
543:82þ in Figs. 4B, C), which was then selected for
tandem MS analysis. The result is shown in Fig. 4C, and
indicates the presence of a y 00 -ion series (i.e., collision-
induced fragment ions that all have the C-terminal res-
idue in common, but differ in length at their N termini).
The mass differences between successive y 00 ions equals
268 G. Sebastiaan Winkler et al. / Methods 26 (2002) 260–269

Fig. 4. Analysis of Elongator ‘‘p30’’ protein composition by multimode mass spectrometry. The gel-bound protein was digested and peptides were
prepared for mass analysis as described in the text. (A) MALDI-reTOF mass spectrum of the 30% peptide pool. Major peaks corresponding to m=z
values that could be matched to predicted tryptic peptides from yeast protein YMR312w are marked with filled squares, those matching ribosomal
protein S4A with closed circles, and those matching ribosomal protein S1B with closed triangles. The ion (m=z ¼ 1086:53) indicated with the arrow
corresponds to the peptide ‘‘DVTGSLHVCR.’’ (B) Continuous-flow (CF) NanoESI single-quadrupole scan of the same 30% pool. (C) CF NanoESI
MS/MS of a doubly charged precursor ion, labeled ‘‘543:82þ ’’ in (B) and (C) (and corresponding to the singly charged ion labeled ‘‘1086.53’’ in (A)).
A limited y 00 -ion series is indicated, plus the corresponding peptide sequence that could be deduced. Note that the sequence reads backward. Ex-
perimental: MALDI-reTOF MS analysis was as described under Fig. 3. ESI-MS (and MS/MS) was done on an API 300 triple-quadrupole instrument
(Applied Biosystems/MDS-SCIEX, Thornhill, Canada), modified with a continuous-flow NanoES source as described [33]. Needle voltage ranged
from 600 to 1000 V; the voltages for the orifice and the curtain plate were set at 5 and 350 V, respectively. Q1 scans were collected using a 0.5-amu
step size and a 3-ms dwell time over a mass range from 400 to 1400 amu. Scans were averaged for statistical analysis, and Q1 resolution was set such
that the charge state of singly, doubly, and triply charged ions could be ascertained. For operation in the MS/MS mode, Q1 was set to transmit the
complete isotopic envelope of the parent. All spectra were averaged with a 0.5-Da step size and a 3-ms dwell time for 5 min over the mass range of the
singly charged m=z. Q3 resolution was set such that the charge state of the fragment ions could be distinguished. Collision energies, as well as CAD
gas pressures, were optimized individually for each peptide so as to obtain the best MS/MS. Spectra were inspected for uninterrupted y 00 -ion series
using the ‘‘find higher AAs’’ routine of the BioToolbox (PE-SCIEX) software. This limited information was taken (together with precursor ion mass)
to search the NR database using the PepFrag protein identification program from the PROWL resource available over the Internet (http://
prowl.rockefeller.edu/PROWL/pepfragch.html). Any protein identification thus obtained was verified by comparing the computer-generated frag-
ment ion series of the predicted tryptic peptide with the experimental MS/MS data.

the molecular mass of individual amino acids, enabling 3. Concluding remarks

us to read limited sequence (indicated in Fig. 4C). Please
note that because of the C terminal anchor point, the
sequence reads backward (right to left). The outcome of
the analysis did not leave any doubt about sequence and
identity of peptide ‘‘543:82þ ,’’ thus confirming the ten-
tative identification of yeast protein YMR312w. This
routine was then repeated for each of the other two
predicted proteins (ribosomal proteins S4A and S1B) as
well, excluding these candidates.
The procedure as outlined here is a generic strategy to
isolate novel protein complexes and identify the indi-
vidual components by mass spectrometry. This method
has been successfully employed with several different
target proteins. The HA and histidine tags need not be on
the same protein, allowing the isolation of different
complexes with common subunits. For proteins prepared
in this manner, especially yeast proteins, mass spectro-
 G. Sebastiaan Winkler et al. / Methods 26 (2002) 260–269
metric identification should be relatively straightforward
in most cases, and with good success, provided some
basic rules are followed. This comes down primarily to
avoiding the three major ‘‘No’s’’ of protein and peptide
nanosample handling: no introduction of keratin (e.g.,
fingerprints, hair) at any stage; no large volumes any-
where, beginning with loading of the gels (one lane only);
no overstaining with silver and/or staining with ‘‘bad’’
silver. By doing so, we have identified hundreds of pro-
teins from many complexes, without the aid of any ro-
botic devices or automated data acquisition and analysis
schemes. During the past 5 years, more than 10 tran-
scription-related complexes were thus successfully dis-
sected using these procedures, leading to the discovery or
new functional assignments of 130 proteins (see Refs.
[6,7,9,20–32]). This is in addition to the 17 single factors
involved in transcriptional regulation (published else-
where) and more than 100 proteins from various tran-
scription complexes currently under study.
As many complexes, or ‘‘functional modules,’’ of
proteins that govern gene expression can be predicted to
exist, experimental approaches of affinity capture and
dissection by mass spectrometry, as described here, will
likely become standard procedures in the transcription
field in the near future.
Acknowledgments
Work in our laboratories was supported by grants
from the Imperial Cancer Research Fund and the Hu-
man Frontier Science Program Project RG0193/97 (to
J.Q.S.) and by NCI Core Grant P30 CA08748 (to P.T.).
G.S.W. was supported by an EMBO Long Term
Fellowship.
References
[1] J. Field, J. Nikawa, D. Broek, B. MacDonald, L. Rodgers, I.A.
Wilson, R.A. Lerner, M. Wigler, Mol. Cell. Biol. 8 (1988) 2159–
2165.
[2] R. Janknecht, G. de Martynoff, J. Lou, R.A. Hipskind, A.
Nordheim, H.G. Stunnenberg, Proc. Natl. Acad. Sci. USA 88
(1991) 8972–8976.
[3] J.Q. Svejstrup, W.J. Feaver, J. LaPointe, R.D. Kornberg, J. Biol.
Chem. 269 (1994) 28044–28048.
[4] G.S. Winkler, W. Vermeulen, F. Coin, J.M. Egly, J.H.J. Hoeij-
makers, G. Weeda, J. Biol. Chem. 273 (1998) 1092–1098.
[5] C.M. Green, H. Erdjument-Bromage, P. Tempst, N.F. Lowndes,
Curr. Biol. 10 (2000) 39–42.
[6] S. Chavez, T. Beilharz, A.G. Rondon, H. Erdjument-Bromage, P.
Tempst, J.Q. Svejstrup, T. Lithgow, A. Aguilera, EMBO J. 19
(2000) 5824–5834.
[7] G.S. Winkler, T.G. Petrakis, S. Ethelberg, M. ToKunaga, H.
Erdjument-Bromage, P. Tempst, J.Q. Svejstrup, J. Biol. Chem.
276 (2001) 32743–32749.
269
[8] E.W. Jones, Genetics 85 (1977) 23–33.
[9] G. Otero, J. Fellows, Y. Li, T. de Bizemont, A.M. Dirac, C.M.
Gustafsson, H. Erdjument-Bromage, P. Tempst, J.Q. Svejstrup,
Mol. Cell 3 (1999) 109–118.
[10] H. Erdjument-Bromage, M. Lui, D.M. Sabatini, S.H. Snyder, P.
Tempst, Protein Sci. 3 (1994) 2435–2446.
[11] M. Mann, P. Højrup, P. Roepstorff, Biol. Mass Spectrom. 22
(1993) 338–345.
[12] D.J.C. Pappin, P. Højrup, A.J. Bleasby, Curr. Biol. 3 (1993) 327–
332.
[13] G. Neubauer, A. King, J. Rappsilber, C. Calvio, M. Watson, P.
Ajuh, J. Sleeman, A. Lamond, M. Mann, Nat. Genet. 20 (1998)
46–50.
[14] J.K. Eng, A.L. McCormack, J.R. Yates III, J. Am. Soc. Mass
Spectrom. 5 (1994) 976–989.
[15] M. Mann, M. Wilm, Anal. Chem. 66 (1994) 4390–4399.
[16] M. Lui, P. Tempst, H. Erdjument-Bromage, Anal. Biochem. 241
(1996) 156–166.
[17] A. Shevchenko, M. Wilm, O. Vorm, M. Mann, Anal. Chem. 68
(1996) 850–858.
[18] F. Gharahdaghi, C.R. Weinberg, D.A. Meagher, B.S. Imai, S.M.
Mische, Electrophoresis 20 (1999) 601–605.
[19] H. Erdjument-Bromage, M. Lui, L. Lacomis, A. Grewal, R.S.
Annan, D.E. MacNulty, S.A. Carr, P. Tempst, J. Chromatogr.
826 (1998) 167–181.
[20] B.R. Cairns, Y. Lorch, Y. Li, M. Zhang, L. Lacomis, H.
Erdjument-Bromage, P. Tempst, J. Du, B. Laurent, R.D. Korn-
berg, Cell 87 (1996) 1249–1260.
[21] B.Ø. Wittschieben, G. Otero, T. de Bizemont, J. Fellows, H.
Erdjument-Bromage, R. Ohba, Y. Li, C.D. Allis, P. Tempst, J.Q.
Svejstrup, Mol. Cell 4 (1999) 123–128.
[22] J. Fellows, H. Erdjument-Bromage, P. Tempst, J.Q. Svejstrup, J.
Biol. Chem. 275 (2000) 12896–12899.
[23] L.C. Myers, C.M. Gustafsson, D.A. Bushnell, M. Lui, H.
Erdjument-Bromage, P. Tempst, R.D. Kornberg, Genes Dev. 12
(1998) 45–54.
[24] C.M. Gustafsson, L.C. Myers, J. Beve, H. Sp ahr, M. Lui, H.
Erdjument-Bromage, P. Tempst, R.D. Kornberg, J. Biol. Chem.
273 (1998) 30851–30854.
[25] Y.W. Jiang, P. Veschambre, H. Erdjument-Bromage, P. Tempst,
J.W. Conaway, R.C. Conaway, R.D. Kornberg, Proc. Natl. Acad.
Sci. USA 95 (1998) 8538–8543.
[26] C. Rachez, B.D. Lemon, Z. Suldan, V. Bromleigh, M. Gamble,
A.M. N€a€ar, H. Erdjument-Bromage, P. Tempst, L.P. Freedman,
Nature 398 (1999) 824–828.
[27] Y. Zhang, R. Iratni, H. Erdjument-Bromage, P. Tempst, D.
Reinberg, Cell 89 (1997) 357–364.
[28] Y. Zhang, Z.-W. Sun, R. Iratni, H. Erdjument-Bromage,
P. Tempst, M. Hampsey, D. Reinberg, Mol. Cell 1 (1998) 1021–
1031.
[29] H.-H. Ng, Y. Zhang, B. Hendrich, C.A. Johnson, B.M. Turner,
H. Erdjument-Bromage, P. Tempst, D. Reinberg, A. Bird, Nat.
Genet. 23 (1999) 58–61.
[30] Y. Zhang, H.-H. Ng, H. Erdjument-Bromage, P. Tempst, A. Bird,
D. Reinberg, Genes Dev. 13 (1999) 1924–1935.
[31] D.W. O’Neill, S.S. Schoetz, R.A. Lopez, M. Castle, L. Rabino-
witz, E. Shor, D. Krawchuk, M.G. Goll, M. Renz, H.P. Seelig, S.
Han, R.H. Seong, S.D. Park, T. Agalioti, N. Munshi, D. Thanos,
H. Erdjument-Bromage, P. Tempst, A. Bank, Mol. Cell. Biol. 20
(2000) 7572–7582.
[32] A.J. Saurin, Z. Shao, H. Erdjument-Bromage, P. Tempst, R.E.
Kingston, Nature 412 (2001) 655–660.
[33] S. Geromanos, G. Freckleton, P. Tempst, Anal. Chem. 72 (2000)
777–790.