Dual modification of Alzheimer's disease PHF-tau protein by lysine methylation and ubiquitylation: a mass spectrometry approach.

Abstract

In sporadic Alzheimer's disease (AD), neurofibrillary lesion formation is preceded by extensive post-translational modification of the microtubule associated protein tau. To identify the modification signature associated with tau lesion formation at single amino acid resolution, immunopurified paired helical filaments were isolated from AD brain and subjected to nanoflow liquid chromatography-tandem mass spectrometry analysis. The resulting spectra identified monomethylation of lysine residues as a new tau modification. The methyl-lysine was distributed among seven residues located in the projection and microtubule binding repeat regions of tau protein, with one site, K254, being a substrate for a competing lysine modification, ubiquitylation. To characterize methyl lysine content in intact tissue, hippocampal sections prepared from post mortem late-stage AD cases were subjected to double-label confocal fluorescence microscopy using anti-tau and anti-methyl lysine antibodies. Anti-methyl lysine immunoreactivity colocalized with 78 ± 13% of neurofibrillary tangles in these specimens. Together these data provide the first evidence that tau in neurofibrillary lesions is post-translationally modified by lysine methylation.

Full text

Page 1

ORIGINAL PAPER
Dual modification of Alzheimer’s disease PHF-tau protein
by lysine methylation and ubiquitylation: a mass spectrometry
approach
Stefani N. Thomas•Kristen E. Funk•
Yunhu Wan•Zhongping Liao•Peter Davies•
Jeff Kuret•Austin J. Yang
Received: 24 June 2011/Revised: 13 October 2011/Accepted: 13 October 2011/Published online: 28 October 2011
? The Author(s) 2011. This article is published with open access at Springerlink.com
Abstract
fibrillary lesion formation is preceded by extensive post-
translational modification of the microtubule associated
protein tau. To identify the modification signature associ-
ated with tau lesion formation at single amino acid
resolution, immunopurified paired helical filaments were
isolated from AD brain and subjected to nanoflow liquid
chromatography–tandem mass spectrometry analysis. The
resulting spectra identified monomethylation of lysine
residues as a new tau modification. The methyl-lysine was
distributed among seven residues located in the projection
In sporadic Alzheimer’s disease (AD), neuro-
and microtubule binding repeat regions of tau protein, with
one site, K254, being a substrate for a competing lysine
modification, ubiquitylation. To characterize methyl lysine
content in intact tissue, hippocampal sections prepared
from post mortem late-stage AD cases were subjected to
double-label confocal fluorescence microscopy using anti-
tau and anti-methyl lysine antibodies. Anti-methyl lysine
immunoreactivity colocalized with 78 ± 13% of neurofi-
brillary tangles in these specimens. Together these data
provide the first evidence that tau in neurofibrillary lesions
is post-translationally modified by lysine methylation.
Keywords
Paired helical filaments ? Tau ? Mass spectrometry ?
Methylation ? Ubiquitylation ? Phosphorylation
Alzheimer’s disease ? Neurofibrillary tangles ?
Introduction
Alzheimer’s disease (AD) is defined in part by the
appearance of intracellular inclusions composed of the
microtubule associated protein tau [35]. The mechanisms
that drive tau lesion formation in the highly prevalent
sporadic form of AD are not fully understood, but appear to
involve abnormal post-translational modifications (PTMs)
that influence tau function, stability, and aggregation pro-
pensity [45]. For example, hyperphosphorylation of tau
protein on certain hydroxy amino acids favors lesion for-
mation by dissociating tau from its microtubule binding
partner [4, 7] and by directly raising its rate and extent of
aggregation [1, 9, 48]. Although tau phosphorylation state
is mediated directly by phosphotransferases, it also is
modulated by competing modifications on hydroxy amino
acids such as O-linked b-N-acetylglucosaminylation (O-
GlcNAcylation) [40]. The reciprocal relationship between
S.N. Thomas and K.E. Funk contributed equally to this work.
Electronic supplementary material
article (doi:10.1007/s00401-011-0893-0) contains supplementary
material, which is available to authorized users.
The online version of this
S. N. Thomas ? A. J. Yang (&)
Greenebaum Cancer Center, University of Maryland,
Baltimore, MD 21201, USA
e-mail: ayang@som.umaryland.edu
K. E. Funk ? J. Kuret (&)
Department of Molecular and Cellular Biochemistry,
The Ohio State University College of Medicine,
Columbus, OH 43210, USA
e-mail: kuret.3@osu.edu
Y. Wan
Department of Epidemiology and Public Health,
University of Maryland, Baltimore, MD, USA
Z. Liao
Molecular and Cellular Cancer Biology Program,
University of Maryland, Baltimore, MD, USA
P. Davies
Department of Pathology and Neuroscience,
Albert Einstein College of Medicine, New York, NY, USA
123
Acta Neuropathol (2012) 123:105–117
DOI 10.1007/s00401-011-0893-0

Page 2

these tau modifications is leveraged by O-GlcNAcase
inhibitors, which by increasing O-GlcNAcylation, lower
phosphorylation stoichiometry and depress neurofibrillary
lesion formation [65]. In addition to hydroxy amino acids,
Lys residues are modified on tau protein, and these too can
influence tau metabolism and aggregation. For example,
ubiquitylation of tau at Lys residues modulates intracellular
tau levels [53, 61], the magnitude of which affects both
nucleation and extension phases of the aggregation reaction
[12]. Together these observations suggest that tau aggre-
gation is under complex regulatory control that involves
crosstalk among diverse and sometimes competing PTMs.
To gain insight into the tau PTM signature most closely
associated with neurofibrillary lesion formation at single
amino acid resolution, we have begun mapping modifica-
tions on authentic, paired helical filaments (PHFs) isolated
from AD brain using mass spectrometry methods, with
special emphasis on Lys modifications [13]. Preliminary
analysis identified K254, K311, and K353 within the tau
microtubule binding repeat region as ubiquitylation sites
that were at least partially occupied in PHFs [13]. Recently,
acetylation was discovered as another Lys-directed tau
modification associated with tau-bearing lesions in AD and
frontotemporal dementia [11, 46]. Tau acetylated in vitro
resulted in occupation of diverse sites that overlapped with
those we had determined to be ubiquitylated in disease
[46]. Therefore, acetylation is another candidate modifi-
cation for regulating tau turnover indirectly through the
ubiquitin–proteasome system [46]. Moreover, in other
biochemical pathways, such as histone-mediated control of
gene expression, certain acetylated Lys residues can
alternatively be methylated, contributing to complex cross-
talk among Lys and hydroxyl amino acid modifications
[36]. These observations suggest that the web of tau PTMs
is potentially complex, with Lys-directed modifications
playing key regulatory roles with respect to rates of tau
turnover and aggregation.
Here we extend our characterization of PHF-tau using
mass spectrometry methods by expanding search criteria to
include both acetyl- and methyl-lysine modifications.
Although we found no evidence for tau acetylation/tri-
methylationatthelevelofdetectionavailableinourdatasets,
the results show that Lys monomethylation is a widespread
PHF-taumodification.Thesedatarevealtaumethylationasa
new tau PTM accompanying PHF deposition in vivo.
Materials and methods
Subjects and tissue preparation
This study used only archival, de-identified post mortem
brain tissue samples from autopsies performed with
informed consent of each patient or relative via procedures
approved by the relevant institutional committees (Uni-
versity of Rochester, USA). Paraformaldehyde-fixed brain
tissue was obtained from six elderly subjects with a clinical
diagnosis of AD [mean age 80 ± 10 years (SD)] that was
confirmed on neuropathological evaluation in which the
Consortium to Establish a Registry for AD (CERAD) age-
adjusted criteria were met [47], and from four pathologi-
cally normal controls [mean age 62 ± 10 years (SD)]
without history of neurologic or psychiatric disorders. All
AD cases satisfied criteria for Braak stages V or VI,
whereas none of the control cases met pathological criteria
for AD [6].
Affinity purification of PHF-tau and enzymatic
digestion
PHF-tau was isolated from pooled, late-stage (Braak stages
V or VI) AD neocortical regions by immunoaffinity chro-
matography (MC1 monoclonal antibody) as described
previously [30]. PHF-tau was digested in solution in the
presence of 40% methanol with either trypsin (Promega) or
Lys-C (Sigma) followed by phosphopeptide enrichment
(Immobilized gallium, Thermo Fisher Scientific) as
detailed earlier [13].
Reductive methylation of recombinant human 4R tau
Reductive methylation was done as described previously
withminormodifications
recombinant full-length human 2N4R tau (100 lg) was re-
suspended in 100 ll of 0.1 M citrate buffer (pH 6) and
methylated (room temperature for 2 h) in the presence of
0.1 M sodium cyanoborohydride and 20 mM formalde-
hyde. Reaction products were separated from reactants by
spin dialysis in 100 mM ammonium bicarbonate, pH 7.8.
[18].Briefly, lyophilized
Liquid chromatography–tandem mass spectrometry
(LC–MS/MS)
Mass spectrometric analysis of PHF-tau was performed
using an LTQ ion trap mass spectrometer controlled by
Xcalibur v.1.4 software (Thermo Electron) coupled online
to a nanoflow XTreme Simple LC system (CVC Micro-
Tech) as previously described [13]. Briefly, peptides were
either loaded onto a trap column (Agilent Zorbax C18
guard column, or Michrom Bioresources peptide cap trap)
or loaded directly into the sample loop with 95% solvent A
(2% acetonitrile, 0.1% formic acid) and 5% solvent B (95%
acetonitrile, 0.1% formic acid). A 60 min linear gradient of
5–25% solvent B was used to elute the peptides from the
reverse phase column (150 mm 9 75 lm, 5 lm 300 A˚
C18; CVC Micro-Tech).
106Acta Neuropathol (2012) 123:105–117
123

Page 3

The mass spectrometer was equipped with a nanospray
ionization source (Thermo Electron) using an uncoated
10 lm i.d. SilicaTip PicoTip nanospray emitter (New
Objective). The spray voltage of the mass spectrometer was
2.0 kV and the heated capillary temperature was 200?C.
The top five ions in each MS1 scan were selected for MS/
MS fragmentation. After ions were selected for MS/MS
fragmentation twice within 30 s, they were dynamically
excluded for 30 s. An MS3 scan was triggered if, among
the three most abundant ions in the MS/MS scan, a neutral
loss of 98, 49, or 32.7 Da (corresponding to a loss of
H3PO4from 1?, 2?, or 3? precursor ions, respectively)
was detected. Other mass spectrometric data generation
parameters were as follows: collision energy 24% (35% for
MS3 scans), MS scan range 400–1,800 m/z, minimum MS
signal intensity 500 counts, minimum MS/MS signal
intensity 100 counts, and MS/MS activation time 120 ms
(30 ms for MS3 scans).
Analysis of mass spectrometric data
Spectra were searched against a UniProtKB human pro-
tein database (version Oct 5, 2010; 20,259 reviewed
sequences; 75,498 non-reviewed sequences) using Bio-
works 3.3.1 SP1 with the SEQUEST algorithm. Search
parameters included 1.5 amu peptide mass tolerance, 1.0
amu fragment tolerance, static Cys ?57 (carbamidome-
thylation) modification and the following differential
modifications: Met ?16 (oxidation); Ser, Thr, Tyr ?80
(phosphorylation); Ser, Thr -18 (dehydroalanine and
2-amino-dehydrobutyric acid, respectively); Lys, Asp, Glu
?14 (monomethylation); Lys ?28 (dimethylation); Lys
?42 (trimethylation/acetylation); and Lys ?114 (ubiqui-
tylation). Fully enzymatic (trypsin or Lys-C) peptides
with up to two missed cleavages and charge-state
dependent cross correlation (XCorr) scores C1.5, 2.5, and
3.0 for 1?, 2?, and 3? peptides, respectively, and DCn
[0.1 were considered as initial positive identifications.
All MS/MS and MS3 spectra of identified post-transla-
tionally modified peptides from the initial screening were
subjected to manual verification.
All raw data from this study are freely available to the
research community on our laboratory’s website, http://
www.proteomeumb.org, which also serves as a data-shar-
ing portal. The original raw data are also available from
http://proteomecommons.org with the following Hash IDs:
Tau trypsin digestion dataset: (Y297YWVSnnKcaY3jSn
QkuCWj7tA1mls59uBBjjBpyqF5hOQ4lSmAuWNhvtdC1
EQCsj5A7 XFM0/3zj9YvVUIJlIuuwcoAAAAAAAABrA==);
Tau Lys-C digestion dataset: (pNmgHCue Fwyw3vtAgIdK
GatW5G8weUl7ArA/Fg?OlChNsahHADGSEQp7iUAOz
ouc81DMfWpeq7Ii1pDcjOkrs1h9BHAAAAAAAAAB
pg==).
Antibodies
Anti-tau mouse monoclonal antibodies Tau5 [43] and AT8
[22] were obtained from Dr. L. I. Binder (Northwestern
University Medical School) and Endogen (Woburn, MA),
respectively. Rabbit polyclonal anti-methyl lysine (meK)
antibody was obtained from Enzo Life Sciences (ADI-
KAP-TF121; Plymouth Meeting, USA). Cy3-conjugated
goat anti-rabbit IgG and Alexa Fluor 488-conjugated goat
anti-mouse secondary antibodies were from Jackson
Immuno Research Laboratories, Inc (West Grove, USA)
and Invitrogen (Carlsbad, USA), respectively. Horseradish
peroxidase-conjugated goat anti-rabbit IgG secondary
antibody used for Western Blot was from Kirkegaard and
Perry Laboratories Inc (Gaithersburg, USA).
Immunohistochemistry
Coronal hippocampal tissue sections were cut (20 lm
thickness) and processed for immunohistochemistry as
described previously [17, 33]. Sections were rehydrated in
PBST (2.7 mM KCl, 0.14 M NaCl, 8.1 mM Na2HPO4,
0.1% Tween-20, pH 7.4) and fixed (10 min) in ice cold
methanol. After 3 9 5 min rinses in PBST, sections were
blocked (1 h at 19?C) with 5% goat serum diluted in PBST,
then incubated (overnight at 4?C) with primary antibodies
diluted in 2.5% goat serum (Tau5, 1 lg/ml; Anti-meK,
0.3 lg/ml; AT8, 0.1 lg/ml). After washing in PBST
(3 9 10 min), sections were incubated (1 h at 19?C) with
fluorescent dye-labeled secondary antibodies (1.5 lg/ml
Cy3-conjugated goat anti-rabbit IgG; 2 lg/ml Alexa Fluor
488-conjugated goat anti-mouse IgG). After washing in
PBST (3 9 5 min), tissue was treated (10 min at 19?C)
with 0.1% Sudan Black B (EM Diagnostics, Gibbstown,
USA) in 80% ethanol to suppress lipofuscin autofluores-
cence [59]. Sections were then washed (2 9 5 min) in
PBST and once for 5 min in 33 mM NaH2PO4,162 mM
Na2HPO4, pH 7.4. Coverslips were mounted with Vecta-
shield (Vector Laboratories, Burlingame, USA) and sealed
with clear nail enamel. Labeled sections were viewed in a
Leica TCS SL laser-scanning confocal system (409 oil
HCX Plan Apo CS 0.75–1.25 NA or 1009 oil HCX Plan
Apo CS 0.70–1.40 NA objective lens) operated at wave-
lengths optimized for simultaneous detection of Alexa
Fluor 488 (kex= 488 nm; kem= 500–530 nm), and Cy3
(kex= 543 nm; kem= 560–600 nm). Long wavelength
fluorescence (kex= 633 nm; kem= 650–740 nm) also
was monitored to assess autofluorescence intensity. Digital
confocal images were captured at 19 and 39 digital zoom,
and stored in Tagged Image Format. Both secondary
antibodies displayed minimal non-specific staining under
these conditions as determined by immunostaining in the
absence of primary antibodies. For immunoadsorption
Acta Neuropathol (2012) 123:105–117 107
123

Page 4

assay, primary antibodies anti-meK (0.3 lg/ml) and AT8
(0.1 lg/ml) were diluted in 2.5% goat serum containing
2000-foldmolar excessof
(0.52 mg/ml) and incubated overnight (4?C) with agitation.
Pellets were separated by centrifugation at 30,0009g for
30 min, and the supernatant was used for immunohisto-
chemistry as described above.
recombinant 2N4Rtau
Analytical methods
The proportion of meK-positive lesions was estimated
using the Wilson score method [17, 49]. Sufficient tech-
nical replicates (defined as Tau-positive bodies at least
7 lm in both length and width) were counted from at least
five fields of each case so that the Wilson 95% confidence
interval for colocalization was\15%. Overall mean colo-
calization was then calculated as the average of all six
biologicalreplicatemeans
deviation.
andreported ± standard
Results
PHF-tau is methylated in its N-terminal projection
and microtubule binding domains
To identify sites of Lys modification in PHF-tau, previ-
ouslycollectedMSdatasets
independent preparations of authentic AD-brain derived
PHFs (digested with either trypsin or Lys-C proteases;
[13]) were interrogated using the SEQUEST database
search algorithm programmed to identify unmodified Lys
residues along with sites of monomethylation (K ? 14),
dimethylation(K ? 28),
obtained fromtwo
trimethylation(K ? 42),
acetylation (also K ? 42), and ubiquitylation (K ? 114).
Sequence coverage in these datasets included 25 out of the
44 Lys residues present in the longest form of human brain
tau protein (2N4R tau, Fig. 1). This sequence coverage is
based on only the unmodified, lysine-modified (methylated
and ubiquitylated) and phosphorylated forms of PHF-tau.
Although these search conditions confirmed three sites of
ubiquitylation in the microtubule binding repeat region
(K254, K311, and K353; [13]), no evidence for either
K ? 28 or K ? 42 masses was found, indicating that
dimethyl-, trimethyl-, and acetyl-lysine were not present in
the coverage area at the level of detection available in our
datasets. However, robust monomethylation was identified
at seven sites distributed throughout the tau sequence
(Table 1). Three of the sites (K163, K174, and K180)
reside within the proline-rich region of the tau N-terminal
projection domain, which mediates interactions with
microtubule-associated proteins such as actin [27] and the
Src homology three domain of plasma membrane-associ-
ated proteins including Src family kinases [37] and
phospholipase Cc [54]. In contrast, K254, K267, and K290
are part of the first and second repeats of the microtubule-
binding domain. They reside within or adjacent to the core
b-sheet region of filamentous tau in vivo [50] and flank the
‘‘PHF6*’’ sequence that modulates fibrillation rate of
recombinant monomeric 4R tau in vitro [29, 39, 64].
Together these data reveal that PHF-tau is monomethylated
in vivo, and that the major occupied sites distribute across
protein segments known to mediate tau-protein interactions
(including tubulin binding and self association).
To assess the relative abundances of methylated, ubiq-
uitylated, and unmodified PHF-tau peptides, data were
subjected to spectral counting, which measures the number
of times a peptide is identified by MS/MS. Because spectral
Fig. 1 Summary of modification sites identified by LC–MS/MS on
immunopurified PHF-tau. The sequence shown is that of human
2N4R tau (NCBI accession number NP_005901). Bold sequence
coverage; Red PHF6 and PHF6* motifs; dashed lines segments
encoded by alternatively spliced exons 2, 3, and 10; underline repeat
region (as defined in [23]); P Phosphorylated sites, U Ubiquitylated
sites, me1 monomethylated sites identified from MS analysis reported
herein and in [13]
108Acta Neuropathol (2012) 123:105–117
123

Page 5

counts correlate linearly with protein abundance [41], they
have been employed for relative quantification in many
label-free proteomic studies [10, 51, 56, 57, 66]. Relative
abundance was calculated by dividing the spectral count of
a modified peptide by the sum of the spectral counts of all
its forms (i.e., modified and unmodified). Results showed
that the relative abundance of monomethylation varied
among sites, from12% (K290) to as high as 67% (K180 and
K267) (Fig. 2). The relative abundance of ubiquitylation
also varied, from 1% (K254) to 33% (K311) (Fig. 2).
These data indicate that Lys modification occupancies are
substantial in PHF tau.
Lys methylation participates in competitive crosstalk
with ubiquitylation
The sites of monomethylation identified above directly
overlapped with sites previously identified as being acet-
ylated in vitro (K174, K180; [46]) or ubiquitylated in vivo
(K254; [13]), raising the issue of competitive crosstalk at
these residues. Although no Lys acetylation was detected at
these sites in our datasets, it was possible to quantify rel-
ative methylation and ubiquitylation of K254. Methylated
K254 was identified in Lys-C peptide aa241-254 from
manually verified MS/MS spectra (Fig. 3). Relative to the
1 ? charge state y-ions of unmodified aa241–254 peptide
(Fig. 3a), the y-ions of methylated aa241–254 were shifted
by ?14 Da (Fig. 3b), reflecting the mass of the methyl
group (CH3) added to (and the H atom lost from) the e-
amino group on the K254 side chain. The appearance of the
?14 Da shift was not likely an artifact of Fischer esterifi-
cation (i.e., chemical methylation of free carboxylic acid
groups in Asp and Glu; [32, 44]), because all MS/MS
spectra were unambiguously assigned by SEQUEST
(examples are shown in Supplemental Table 1). Moreover,
care was taken to limit Fischer esterification conditions
during sample preparation by maintaining the pH of
digestion buffers [8, by avoiding exposure of samples to
high concentrations of methanol in the presence of acetic
acid, and by avoiding methanol as a co-solvent during LC
separation of the peptides.
Ubiquitylated-K254 was identified in tryptic peptide
aa243–257, which was resistant to protease cleavage at
K254 owing to the isopeptide bond formed between
ubiquitin and K254 (Fig. 3c). When aa241–254 and aa243–
257 peptides were subjected to spectral counting, 41% of
aa241–254 was monomethylated, whereas only1% of
aa243–257 was ubiquitylated (Table 2; Fig. 2). These data
Table 1 Lys methylation sites identified on PHF-tau
aa ResiduesPeptide Methyl siteCharge state(s) PHF-tau domainXCorr score
D Cn
25–44 DQGGYTMHQDQEGDTDAGLK
K443?N4.070.16
151–163IATPRGAAPPGQK
K1632?N, P3.28 0.42
164–174 GQANATRIPAK
K174 2?N, P3.030.28
175–180 TPPAPK
K1801? N, P 1.590.33
241–254SRLQTAPVPMPDLK
K2542?/3? M, R1 3.71/3.550.41/0.24
258–267SKIGSTENLK
K2672?M, R1 3.14 0.40
281–290KLDLSNVQSK
K2902?M, R23.56 0.28
Lys-C and trypsin in-solution digests of PHF-tau were analyzed by nanoflow LC–MS/MS and the data were searched against a human database
using Bioworks with the SEQUEST algorithm
Amino acids in bold indicate identified methylated Lys residues. Amino acid (aa) residue numbering is based on the human 2N4R tau isoform
(NCBI accession number NP_005901). Cross-correlation (XCorr) and Delta correlation (DCn) scores are two metrics in the SEQUEST algorithm
used to assess the quality of candidate peptides assigned to MS/MS spectra. Peptides with charge-state dependent XCorr scores C1.5, 2.5, and 3.0
for 1?, 2?, and 3? peptides, respectively, and DCn scores[0.1 were the criteria for positive identifications
N N-terminal projection domain, P Pro-rich region, M microtubule-binding domain, R1, R2 repeat regions 1 and 2, respectively
Fig. 2 Relative abundance of PHF-tau methylation and ubiquityla-
tion. Relative abundance was calculated based on the spectral counts
of the modified peptide/(modified peptide ? unmodified peptide).
Only sites having total spectral counts[3 are shown. Inset quantifies
K254 methylation (41%) and ubiquitylation (1%). K254 was the most
abundant methylated site identified on PHF-tau (spectral count = 17)
and the only site on PHF-tau identified in methylated and ubiquity-
lated forms
Acta Neuropathol (2012) 123:105–117109
123

Page 6

suggest that monomethylation competes directly with ub-
iquitylation at K254, and that monomethylation of this site
is far more abundant than ubiquitylation in PHF-tau
(Fig. 2, inset).
Lys methylation and potential cross talk
with phosphorylation
Although several of the monomethylation sites on PHF-tau
lie within five amino acid residues of phosphorylation sites
identifiedintheliterature
(aa258–267) was detected in our data sets that contained
both a methylated lysine and a phosphorylated hydroxy
amino acid. The phosphorylation site in this peptide, S262,
functions as a regulatory gatekeeper for microtubule
association, with site occupancy correlating with decreased
binding affinity [38, 58]. Its proximity to K267 allowed us
to detect relative site occupancy within a short peptide
sequence. Unmodified aa258–267 was identified as a Lys-
C derived peptide from its MS/MS spectrum (Fig. 4a).
Relative to the 1? charge state y-ions of this peptide, the y-
ions of methylated aa258–267 peptide were shifted by
?14 Da, reflecting the mass of the methyl group added to
the e-amino group of K267 (Fig. 4b). In contrast, the
assigned y7- and y8-ions of aa258–267 phosphorylated at
S262 were shifted by -18 Da, consistent with the domi-
nant ion having undergone neutral loss of H3PO4by b-
elimination ([3]; Fig. 4c). Peptide aa258–267 also was
detected in a doubly modified (phosphorylated and meth-
ylated)form.Consistent
phosphopeptides, the MS/MS spectrum of this peptide was
dominated by a precursor ion that had undergone b-elim-
ination (Fig. 4d).
To quantify the modification signature of these peptides,
their relative abundance was measured by spectral counting
(Table 3). Results revealed that the singly modified
methyl-K267 peptide was the most abundant form (spectral
count = 6; 35% of total aa258–267 peptide spectral count),
and relative abundance followed the pattern: methyl-
K267 * methyl-K267/phospho-S262[phospho-
S262 * un-modified K267/S262. Thus monomethylation
at K267 was a more abundant PHF-tau modification than
was phosphorylation at S262, with the latter being found
more frequently in the presence of K267 methylation than
found alone.
[25],only one peptide
withthe behavior of
Methyl-lysine immunoreactivity associates
with the neurofibrillary lesions of AD
In AD, PHF-tau accumulates within neurofibrillary lesions
associated with neuronal cell bodies (NFTs), neuronal
processes (neuropil threads), and the dystrophic neurites of
Fig. 3 MS/MS characterization of K254 modification site. Spectra
for peptide aa241–254 derived from Lys-C cleavage of PHF-tau
identified in (a) unmodified and (b) methylated (asterisks site of
methylation) forms. c Spectrum for tryptic peptide aa243–257
identified in ubiquitylated form (carets site of ubiquitylation). Trypsin
did not cleave ubiquitylated aa243–257 at K254 owing to its
conjugation to the C-terminal di-Gly fragment of ubiquitin
110 Acta Neuropathol (2012) 123:105–117
123

Page 7

Fig. 4 MS/MS identification of S262 phosphorylated and K267
methylated PHF-tau peptides. Spectra for PHF-tau derived peptide
aa258–267 identified in a unmodified, b monomethylated (asterisks
site of methylation), c Ser phosphorylated (at sign site of phosphor-
ylation), and d doubly modified (asterisks site of methylation; at sign
site of phosphorylation) forms
Table 2 Relative abundances of methylated and ubiquitylated PHF-tau peptides as assessed by spectral counts
aa ResiduesPeptideModification siteSpectral count% Modified
ModifiedUn-modifiedTotal
25–44DQGGYTMHQDQEGDTDAGLK
meK44 11182938%
151–163 IATPRGAAPPGQK
meK163268 25%
164–174GQANATRIPAK
meK1742n.d.2 n.d.
175–180TPPAPK
meK180213 67%
241–254 SRLQTAPVPMPDLK
meK25417 2441 41%
243–257LQTAPVPMPDLKNVK ub-K2542188 1901%
258–267SKIGSTENLK
meK267639 67%
281–290 KLDLSNVQSK
meK2902 1416 12%
299–317HVPGGGSVQIVYKPVDLSKub-K3115 10 15 33%
350–369 VQSKIGSLDNITHVPGGGNKub-K3532 4951 4%
Spectral counts are defined as the number of times a peptide was identified by MS/MS. Amino acids in bold indicate identified methylated Lys
residues. % Modified is calculated based on the following spectral counts: modified peptide/(modified peptide ? un-modified peptide)
n.d. not detected, me methylation, ub ubiquitylation
Acta Neuropathol (2012) 123:105–117 111
123

Page 8

neuritic plaques [8]. To determine whether tau methylation
correlated spatially with neurofibrillary pathology, sections
of AD brain hippocampus were probed with a polyclonal
antibody that binds meK-containing proteins along with
well-characterized monoclonal anti-tau antibodies AT8 and
Tau5 [5, 43] in double label format. These tau antibodies
were chosen for analysis because their epitopes are present
during all phases of NFT development, including the early
pre- and intracellular-NFT stages [60]. The specificity of
the anti-meK reagent was first tested on recombinant 2N4R
tau preparations that were subjected to reductive methyl-
ation in vitro (see materials and methods). When subjected
to SDS-PAGE, both non-methylated and methylated 2N4R
tau migrated as single species, with the latter undergoing a
band shift to 73 kDa (Fig. 5a). Rabbit polyclonal anti-meK
antibody strongly labeled this species but not nonmethy-
lated 2N4R tau (Fig. 5b). Binding specificity was further
characterized by preadsorption assay, where antibodies
were preincubated with 2000-fold molar excess of either
non-methylated or methylated 2N4R tau before being used
for immunohistochemical labeling of the hippocampal
brain sections. When AT8 was preadsorbed under these
conditions, neither non-methylated nor methylated tau
preparations diminished its reactivity toward neurofibril-
lary lesions (detected by confocal immunofluorescence
microcopy; Fig. 5c, f). These data were consistent with the
established anti-phosphoepitope binding specificity of AT8
[22]. Anti-meK antibody also labeled neurofibrillary
lesions in these sections, and like AT8, its labeling inten-
sity was not affected by preadsorption with non-methylated
2N4R tau (Fig. 5d). However, labeling intensity was
greatly diminished by pre-adsorption with methylated
2N4R tau (Fig. 5g). Together these data are consistent with
the anti-meK antibody being selective for lysine methyl-
ated proteins.
Double-label confocal immunofluorescence studies were
then extended to hippocampal brain sections prepared from
four cognitively normal and six late-stage AD cases (case
demographics are summarized in Table 4). In normal tis-
sue, AT8, Tau5, and anti-meK antibody staining was
diffuse and rarely colocalized (shown for AT8 and anti-
meK antibody in Fig. 6a–c). In contrast, both AT8
(Fig. 6d–i) and Tau 5 (Fig. 6j–o) strongly labeled neuro-
fibrillary lesions in AD hippocampal sections, including
NFTs, neuropil threads, and neuritic plaques. At high
magnification, NFTs immunolabeled with Tau5 displayed a
fibrillar pattern throughout the lesions (Fig. 6m), whereas
those labeled with AT8 also displayed a pattern of intense
immunoreactivity enriched on the outer rims of the lesions
(Fig. 6g). Rim staining, which has been seen previously
with AT8 [28], was particularly conspicuous at low fluo-
rescence gain. In contrast, anti-meK immunoreactivity
appeared diffusely distributed throughout the sections
(Fig. 6e, h, k, n), with the most intense labeling correlating
with both AT8- and Tau5-stained lesions (Fig. 6d–o). In
particular, the pattern of labeling with NFTs resembled that
of Tau5, with fibrillar staining throughout the lesion
(Fig. 6m–o). Although background fluorescence was gen-
erally too high to detect neuropil threads with the anti-meK
antibody, colocalization with particularly large or intense
dystrophic neurites was occasionally seen (Fig. 6j–l). NFT
labeling was robust, however, and so colocalization of
meK immunoreactivity with this lesion was quantified in
hippocampus CA1 region. Results showed that the majority
of NFTs labeled with anti-meK antibody in all six cases
(Table 4). Overall, anti-meK colocalization with AT8-
labeled NFTs averaged 78 ± 11% (SD, n = 6 cases)
whereas colocalization with Tau5-labeled NFTs averaged
79 ± 16% (SD, n = 6 cases). Together these data show
that the methylation of PHF-tau identified through mass
spectrometry is widespread in this affected brain region,
with most NFTs harboring anti-meK immunoreactivity in
late-stage AD.
Discussion
Although certain familial tauopathies result from missense
mutations in the tau gene (MAPT), AD pathogenesis is
not associated with changes in tau amino acid sequence.
Rather, tau lesion formation in sporadic AD is accom-
panied by PTMs that contribute to pathogenesis by
modulating tau function, stability, and aggregation pro-
pensity. Because neurofibrillary lesion density correlates
with neurodegeneration [24, 31] and cognitive decline
[19, 20, 55], a high priority in the AD field is to identify
the PTM signature that drives neurofibrillary lesion for-
mation in sporadic disease. Here we found that PHF-tau
isolated from neocortical areas is methylated at at least
seven distinct sites, and that the modification is wide-
spread among NFTs in the CA1 region of hippocampus in
late stage AD. These findings suggest that tau methylation
is a component of the PTM signature associated with
PHF-tau.
Table 3 Relative abundance of S262 phosphorylated and K267
methylated PHF-tau peptides (aa258–267)
Peptide (aa258–267) Spectral count% Relative
abundance
Unmodified318
Phospho-S262318
Phospho-S262, meK267529
meK267 635
Relative abundances were calculated by dividing the peptide spectral
count for each peptide of interest by the total count for all peptides
112 Acta Neuropathol (2012) 123:105–117
123

Page 9

Fig. 5 Anti-meK antibody specificity. Aliquots of unmodified (-)
and in vitro methylated (?) 2N4R tau were separated by SDS-PAGE
(8% polyacrylamide) and either a stained with Coomassie Blue
(500 ng tau proteins), or b subjected to immunoblot analysis with
anti-meK antibody (100 ng tau protein). Molecular mass calibration
markers (M) are shown in units of kDa. High-stoichiometry reductive
methylation reduced recombinant 2N4R migration on SDS-PAGE. To
further test specificity, antibodies AT8 (c, f; green channel) and anti-
meK (d, g; red channel) were pre-adsorbed with either unmodified
(c–e) or in vitro methylated (f–h) 2N4R tau, and then subjected to
double-label confocal immunofluorescence microscopy on sections of
AD hippocampus. Lesions labeled with both AT8 and anti-meK are
marked by asterisks. AT8 labeling was unaffected by preadsorption
with either unmodified or methylated tau, whereas anti-meK immu-
noreactivity was diminished by pre-adsorption with methylated (f–h;
carets), but not unmodified tau (c–e)
Table 4 Case demographics and marker colocalization in hippocampus CA1 region
Case (#) Age (years) Gender PMI (h)Diagnosis meK/AT8
colocalization
± SE (%)
n
meK/Tau5
colocalization
± SE (%)
n
1 61M5.3 AD, Braak stage V–VI86 ± 84392 ± 636
2 81F– AD; Braak stage V–VI, vascular involvement89 ± 7 3989 ± 8 36
3 81M 4.1 AD; Braak stage VI, vascular involvement78 ± 6154 82 ± 5 168
4 82F4.0AD, Braak stage V–VI 78 ± 711590 ± 670
5 86M3.2 AD, Braak stage V–VI 59 ± 1270 52 ± 1097
6 87M 2.5 AD, Braak stage V–VI75 ± 1428 71 ± 1335
752M6.5 Control, myocardial infarct––––
8 57M7.5 Control, cardiac failure––––
964M7.0Control ––––
1075M8.2Control, liver failure ––––
PMI post mortem interval, n number of lesions quantified
Acta Neuropathol (2012) 123:105–117113
123

Page 10

The direct effects of Lys methylation on tau function are
not known, but in other proteins this modification is
reported tomodulateprotein–protein interaction
increasing the interaction radius of Lys side chains [34] and
by increasing the entropic driving force for its burial away
from solvent [62]. For these reasons, reductive methylation
is used to improve the performance of protein crystalliza-
tion in preparation for diffraction experiments [34]. The
localization of methylated residues within the microtubule
by
binding repeat region in proximity to the PHF6 and PHF6*
nucleation centers makes Lys methylation a candidate
modification for directly affecting normal tau interactions
with binding partners such as microtubules as well as
abnormal interactions such as aggregation and PHF
formation.
In addition to direct effects, Lys methylation also may
modulate protein function through cross-talk with other
PTMs. In its simplest form, crosstalk involves direct
Fig. 6 Anti-meK
immunoreactivity colocalizes
with neurofibrillary lesions in
AD hippocampus. Double-label
confocal images of hippocampal
sections (CA1 region) stained
with anti-tau mouse monoclonal
antibodies AT8 (a–i; green
channel) or Tau5 (j–o; green
channel) along with the rabbit
anti-meK antibody (red
channel). In AD cases, low
magnification images (409
objective,19 zoom) show
colocalization of anti-meK
immunoreactivity with NFTs
(d–f; asterisks) and a neuritic
plaque (j–l; arrows). High
magnification images (1009
objective, 39 zoom) show
typical morphology of NFTs (g–
i and m–o). Image overlays
highlight pixel overlap between
anti-meK and AT8 (d–i) and
Tau5 (j–o) immunoreactivity.
Anti-meK immunoreactivity
colocalized extensively with
NFTs in all fields examined. In
contrast, AT8 and anti-meK
immunoreactivity did not
colocalize in hippocampal
sections prepared from
cognitively normal cases (a–c)
114 Acta Neuropathol (2012) 123:105–117
123

Page 11

competition for occupancy of any single amino acid resi-
due side chain. Here we found that K254 from PHF-tau
isolated from AD brains was either ubiquitylated or
methylated, with the latter strongly predominating. These
data indicate that K254 methylation must occur at the
expense of ubiquitylation, and thus is a candidate modifi-
cation for regulating protein degradation via the ubiquitin–
proteasome system [46].
A second, more complex paradigm of crosstalk involves
interactions between separate amino acid residues, where
modification of one residue alters the ability of a second
residue to bind its modifying enzyme. For example, tau
phosphorylated by the microtubule affinity-regulating
kinase 2 (PAR1/MARK2) is not recognized by the CHIP/
Hsp90 E3 ligase complex and thus fails to be ubiquitylated
or degraded [15]. Conversely, tau requires phosphorylation
by GSK3b for efficient recognition and ubiquitylation by
the CHIP/Hsp90 complex [61]. Here we found that K267 is
methylated in PHF-tau, and that phosphorylation of S262
appears more frequently on peptides containing methylated
rather than non-methylated K267. This observation raises
the possibility of crosstalk between S262 phosphorylation,
which strongly reduces the binding of tau to microtubules
[4, 58], and K267 methylation. The impaired microtubule-
binding phenotype associated with S262 phosphorylation
may be important for AD pathogenesis since this site is
occupied early in NFT formation [2]. Other sites of phos-
phorylation identified previously in PHF-tau [25] (but not
found in our datasets) also lie within close proximity to
methylation sites. These include T175, T181, S184, S185,
S258, and S289. Moreover, one projection domain meth-
ylation site, K44, is adjacent to a predicted calpain-
catalyzed cleavage site in tau [52]. These data suggest that
tau methylation is positioned to potentially engage in cross
talk with multiple post translational modifications. It will
be important to establish the temporal relationship among
these modifications and the aggregation of tau during
neurofibrillary lesion formation in AD. Mass spectrometry
can complement high throughput immunohistochemical
detection methods, which are sensitive to modification
context [16], in this effort.
Comparison with previous studies
Previous characterization of PHFs prepared by differential
centrifugation using amino acid analysis reported a low
unmodified Lys content relative to predicted tau composi-
tion, consistent with extensive Lys modification, but failed
to detect meK [42]. The source of this discrepancy is not
clear, but may relate to the sensitivity of amino acid
analysis relative to MS and to the purity of the PHF
preparations used for study.
Two recent studies reported that tau can be acetylated on
Lys in vitro, resulting in increased aggregation propensity
and inhibition of tau degradation [11, 46]. In vivo acety-
lation of K163, K174, K180 and K280 and accumulation
within PHF-tau was claimed on the basis of antibody-based
methods in tissue sections prepared from AD and fronto-
temporal lobar degeneration cases [11, 46]. On the basis of
MS analysis, we did not detect tau acetylation even though
residues K163, K174, K180 and K280 were resolved
within our coverage area. These discrepancies could arise
from any of several possibilities. One is that methylation
occurs to a greater extent than acetylation. Consistent with
this model, we found that K163, K174, and K180 were
modified in PHF, but by monomethylation rather than
acetylation. Combined with our detection of Lys ubiqui-
tylation, these data suggest that the relative abundances of
Lys methylation and ubiquitylation are likely higher than
Lys acetylation in the PHF-tau preparation used herein.
Another possibility is that PHF-tau contains substantial
acetylation, but at sites distinct from those identified in
vitro or that reside within our current coverage area.
Additional analysis of PHF will be required to test this
hypothesis. A third possibility is that immunopurified PHF
represents a subfraction of tau aggregates with greater
SDS-solubililty. For example, we find ([13]; Fig. 1) that
immunopurified PHFs lack detectable phosphorylation at
T175 and T181 that were reported qualitatively by others in
some tissue/PHF preparations [2, 21, 26]. In addition, Y394
phosphorylation has been reported in PHF-tau [14, 63], but
this modification is present at rather low relative abundance
in the PHF-tau preparation used herein. It should be pos-
sible to resolve these possibilities in the future using mass
spectrometry under conditions that provide quantitative
information on modification occupancy.
In conclusion, we report biophysical evidence that seven
Lys residues (K44, K163, K174, K180, K254, K267, and
K290) in PHF-tau immunopurified from AD brain are
monomethylated. The sites present opportunities for cross
talk with other PTMs, including direct competition with
ubiquitylation and acetylation, and indirect interaction with
phosphorylation and proteolysis. It will be important to
determine the functional and temporal relationships among
these established modifications in modulating the accu-
mulation of PHF-tau in AD and other age-related
neurodegenerative diseases.
Acknowledgments
of Health Grants MH59786 and AG25323 to A.Y. J.K is supported by
AG14452. Y.W. and S.T. were supported by the Maryland Cigarette
Restitution Fund. The access to the confocal microscope at Ohio State
University was supported by Core Center grant P30-NS045758. The
authors thank Diane Cripps for excellent technical assistance in the
preparation of PHF-tau for LC–MS/MS analysis and also for the
optimization of the LC–MS/MS platform used for the studies
This work was supported by National Institutes
Acta Neuropathol (2012) 123:105–117115
123

Page 12

described herein. We thank Drs. Paul D. Coleman and Linda M.
Callahan (University of Rochester, NY) for generous access to tissue
resources.
Open Access
Creative Commons Attribution Noncommercial License which per-
mits any noncommercial use, distribution, and reproduction in any
medium, provided the original author(s) and source are credited.
This article is distributed under the terms of the
References
1. Alonso A, Zaidi T, Novak M, Grundke-Iqbal I, Iqbal K (2001)
Hyperphosphorylation induces self-assembly of tau into tangles
of paired helical filaments/straight filaments. Proc Natl Acad Sci
USA 98:6923–6928
2. Augustinack JC, Schneider A, Mandelkow EM, Hyman BT
(2002) Specific tau phosphorylation sites correlate with severity
of neuronal cytopathology in Alzheimer’s disease. Acta Neuro-
pathol (Berl) 103:26–35
3. Beausoleil SA, Jedrychowski M, Schwartz D et al (2004) Large-
scale characterization of HeLa cell nuclear phosphoproteins. Proc
Natl Acad Sci USA 101:12130–12135
4. Biernat J, Gustke N, Drewes G, Mandelkow EM, Mandelkow E
(1993) Phosphorylation of Ser262 strongly reduces binding of tau
to microtubules: distinction between PHF-like immunoreactivity
and microtubule binding. Neuron 11:153–163
5. Braak H, Alafuzoff I, Arzberger T, Kretzschmar H, Del Tredici K
(2006) Staging of Alzheimer disease-associated neurofibrillary
pathology using paraffin sections and immunocytochemistry.
Acta Neuropathol 112:389–404
6. Braak H, Braak E (1991) Neuropathological stageing of Alzhei-
mer-related changes. Acta Neuropathol 82:239–259
7. Bramblett GT, Goedert M, Jakes R, Merrick SE, Trojanowski JQ,
Lee VM (1993) Abnormal tau phosphorylation at Ser396 in
Alzheimer’s disease recapitulates development and contributes to
reduced microtubule binding. Neuron 10:1089–1099
8. Buee L, Bussiere T, Buee-Scherrer V, Delacourte A, Hof PR
(2000) Tau protein isoforms, phosphorylation and role in neu-
rodegenerative disorders. Brain Res Brain Res Rev 33:95–130
9. Chang E, Kim S, Schafer KN, Kuret J (2011) Pseudophosph-
orylation of tau protein directly modulates its aggregation
kinetics. Biochim Biophys Acta 1814:388–395
10. Choi H, Fermin D, Nesvizhskii AI (2008) Significance analysis of
spectral count data in label-free shotgun proteomics. Mol Cell
Proteomics 7:2373–2385
11. Cohen TJ, Guo JL, Hurtado DE et al (2011) The acetylation of
tau inhibits its function and promotes pathological tau aggrega-
tion. Nat Commun 2:252
12. Congdon EE, Kim S, Bonchak J, Songrug T, Matzavinos A,
Kuret J (2008) Nucleation-dependent tau filament formation: the
importance of dimerization and an estimation of elementary rate
constants. J Biol Chem 283:13806–13816
13. Cripps D, Thomas SN, Jeng Y, Yang F, Davies P, Yang AJ
(2006) Alzheimer disease-specific conformation of hyperphos-
phorylated paired helical filament-Tau is polyubiquitinated
through Lys-48, Lys-11, and Lys-6 ubiquitin conjugation. J Biol
Chem 281:10825–10838
14. Derkinderen P, Scales TM, Hanger DP et al (2005) Tyrosine 394
is phosphorylated in Alzheimer’s paired helical filament tau and
in fetal tau with c-Abl as the candidate tyrosine kinase. J Neurosci
25:6584–6593
15. Dickey CA, Koren J, Zhang YJ et al (2008) Akt and CHIP
coregulate tau degradation through coordinated interactions. Proc
Natl Acad Sci USA 105:3622–3627
16. Fuchs SM, Krajewski K, Baker RW, Miller VL, Strahl BD (2011)
Influence of combinatorial histone modifications on antibody and
effector protein recognition. Curr Biol 21:53–58
17. Funk KE, Mrak RE, Kuret J (2011) Granulovacuolar degeneration
(GVD) bodies of Alzheimer’s disease (AD) resemble late-stage
autophagic organelles. Neuropathol Appl Neurobiol 37:295–306
18. Geoghegan KF (2001) Modification of amino groups. Curr Protoc
Protein Sci Chapter 15, Unit 15.2
19. Ghoshal N, Garcia-Sierra F, Wuu J et al (2002) Tau conforma-
tional changes correspond to impairments of episodic memory in
mild cognitive impairment and Alzheimer’s disease. Exp Neurol
177:475–493
20. Giannakopoulos P, Herrmann FR, Bussiere T et al (2003) Tangle
and neuron numbers, but not amyloid load, predict cognitive
status in Alzheimer’s disease. Neurology 60:1495–1500
21. Goedert M, Jakes R, Crowther RA et al (1994) Epitope mapping
of monoclonal antibodies to the paired helical filaments of Alz-
heimer’s disease: identification of phosphorylation sites in tau
protein. Biochem J 301(Pt 3):871–877
22. Goedert M, Jakes R, Vanmechelen E (1995) Monoclonal anti-
body AT8 recognises tau protein phosphorylated at both serine
202 and threonine 205. Neurosci Lett 189:167–169
23. Goedert M, Spillantini MG, Potier MC, Ulrich J, Crowther RA
(1989) Cloning and sequencing of the cDNA encoding an isoform
of microtubule-associated protein tau containing four tandem
repeats: differential expression of tau protein mRNAs in human
brain. EMBO J 8:393–399
24. Gomez-Isla T, Price JL, McKeel DW Jr, Morris JC, Growdon JH,
Hyman BT (1996) Profound loss of layer II entorhinal cortex
neurons occurs in very mild Alzheimer’s disease. J Neurosci
16:4491–4500
25. Hanger DP, Anderton BH, Noble W (2009) Tau phosphorylation:
the therapeutic challenge for neurodegenerative disease. Trends
Mol Med 15:112–119
26. Hanger DP, Betts JC, Loviny TL, Blackstock WP, Anderton BH
(1998) New phosphorylation sites identified in hyperphospho-
rylated tau (paired helical filament-tau) from Alzheimer’s disease
brain using nanoelectrospray mass spectrometry. J Neurochem
71:2465–2476
27. He HJ, Wang XS, Pan R, Wang DL, Liu MN, He RQ (2009) The
proline-rich domain of tau plays a role in interactions with actin.
BMC Cell Biol 10:81
28. Ikonomovic MD, Abrahamson EE, Isanski BA et al (2006) X-34
labeling of abnormal protein aggregates during the progression of
Alzheimer’s disease. Methods Enzymol 412:123–144
29. Iliev AI, Ganesan S, Bunt G, Wouters FS (2006) Removal of
pattern-breaking sequences in microtubule binding repeats pro-
duces instantaneous tau aggregation and toxicity. J Biol Chem
281:37195–37204
30. Jicha GA, Bowser R, Kazam IG, Davies P (1997) Alz-50 and
MC-1, a new monoclonal antibody raised to paired helical fila-
ments, recognize conformational epitopes on recombinant tau.
J Neurosci Res 48:128–132
31. Josephs KA, Whitwell JL, Ahmed Z et al (2008) Beta-amyloid
burden is not associated with rates of brain atrophy. Ann Neurol
63:204–212
32. Jung SY, Li Y, Wang Y, Chen Y, Zhao Y, Qin J (2008) Com-
plications in the assignment of 14 and 28 Da mass shift detected
by mass spectrometry as in vivo methylation from endogenous
proteins. Anal Chem 80:1721–1729
33. KannanayakalTJ,TaoH,VandreDD,KuretJ(2006)Caseinkinase-
1 isoforms differentially associate with neurofibrillary and granu-
lovacuolar degeneration lesions. Acta Neuropathol 111:413–421
34. Kim Y, Quartey P, Li H et al (2008) Large-scale evaluation of
protein reductive methylation for improving protein crystalliza-
tion. Nat Methods 5:853–854
116Acta Neuropathol (2012) 123:105–117
123

Page 13

35. Kuret J (2007) Detection and reduction of neurofibrillary lesions.
In: Smith HJ, Sewell RDE, Simons C (eds) Protein folding dis-
eases: enzyme inhibitors and other agents as prospective
therapies. CRC Press, Boca Raton, pp 287–324
36. Latham JA, Dent SY (2007) Cross-regulation of histone modifi-
cations. Nat Struct Mol Biol 14:1017–1024
37. Lee G, Newman ST, Gard DL, Band H, Panchamoorthy G (1998)
Tau interacts with src-family non-receptor tyrosine kinases. J Cell
Sci 111:3167–3177
38. Leger J, Kempf M, Lee G, Brandt R (1997) Conversion of serine
to aspartate imitates phosphorylation-induced changes in the
structure and function of microtubule-associated protein tau.
J Biol Chem 272:8441–8446
39. Li W, Lee VM (2006) Characterization of two VQIXXK motifs
for tau fibrillization in vitro. Biochemistry 45:15692–15701
40. Liu F, Iqbal K, Grundke-Iqbal I, Hart GW, Gong CX (2004)
O-GlcNAcylation regulates phosphorylation of tau: a mechanism
involved in Alzheimer’s disease. Proc Natl Acad Sci USA
101:10804–10809
41. Liu H, Sadygov RG, Yates JR 3rd (2004) A model for random
sampling and estimation of relative protein abundance in shotgun
proteomics. Anal Chem 76:4193–4201
42. Liu WK, Ksiezak-Reding H, Yen SH (1991) Abnormal tau pro-
teins from Alzheimer’s disease brains. Purification and amino
acid analysis. J Biol Chem 266:21723–21727
43. LoPresti P, Szuchet S, Papasozomenos SC, Zinkowski RP, Binder
LI (1995) Functional implications for the microtubule-associated
protein tau: localization in oligodendrocytes. Proc Natl Acad Sci
USA 92:10369–10373
44. Lu A, Zougman A, Pudelko M et al (2009) Mapping of lysine
monomethylation of linker histones in human breast and its
cancer. J Proteome Res 8:4207–4215
45. Martin L, Latypova X, Terro F (2011) Post-translational modi-
fications of tau protein: implications for Alzheimer’s disease.
Neurochem Int 58:458–471
46. Min SW, Cho SH, Zhou Y et al (2010) Acetylation of tau inhibits
its degradation and contributes to tauopathy. Neuron 67:953–966
47. Mirra SS, Heyman A, McKeel D et al (1991) The Consortium to
Establish a Registry for Alzheimer’s Disease (CERAD); Part II.
Standardization of the neuropathologic assessment of Alzhei-
mer’s disease. Neurology 41:479–486
48. Necula M, Kuret J (2004) Pseudophosphorylation and glycation
of tau protein enhance but do not trigger fibrillization in vitro.
J Biol Chem 279:49694–49703
49. Newcombe RG (1998) Improved confidence intervals for the
difference between binomial proportions based on paired data.
Stat Med 17:2635–2650
50. Novak M, Kabat J, Wischik CM (1993) Molecular characteriza-
tion of the minimal protease resistant tau unit of the Alzheimer’s
disease paired helical filament. Embo J 12:365–370
51. Old WM, Meyer-Arendt K, Aveline-Wolf L et al (2005) Com-
parison of label-free methods for quantifying human proteins by
shotgun proteomics. Mol Cell Proteomics 4:1487–1502
52. Park SY, Ferreira A (2005) The generation of a 17 kDa neuro-
toxic fragment: an alternative mechanism by which tau mediates
beta-amyloid-induced neurodegeneration. J Neurosci 25:5365–
5375
53. Petrucelli L, Dickson D, Kehoe K et al (2004) CHIP and Hsp70
regulate tau ubiquitination, degradation and aggregation. Hum
Mol Genet 13:703–714
54. Reynolds CH, Garwood CJ, Wray S et al (2008) Phosphorylation
regulates tau interactions with Src homology 3 domains of
phosphatidylinositol 3-kinase, phospholipase C gamma1, Grb2,
and Src family kinases. J Biol Chem 283:18177–18186
55. Royall DR, Palmer R, Mulroy AR et al (2002) Pathological
determinants of the transition to clinical dementia in Alzheimer’s
disease. Exp Aging Res 28:143–162
56. Sardiu ME, Cai Y, Jin J et al (2008) Probabilistic assembly of
human protein interaction networks from label-free quantitative
proteomics. Proc Natl Acad Sci USA 105:1454–1459
57. Schmidt MW, Houseman A, Ivanov AR, Wolf DA (2007)
Comparative proteomic and transcriptomic profiling of the fission
yeast Schizosaccharomyces pombe. Mol Syst Biol 3:79
58. Schneider A, Biernat J, von Bergen M, Mandelkow E, Mandel-
kow EM (1999) Phosphorylation that detaches tau protein from
microtubules (Ser262, Ser214) also protects it against aggrega-
tion into Alzheimer paired helical filaments. Biochemistry
38:3549–3558
59. Schnell SA, Staines WA, Wessendorf MW (1999) Reduction of
lipofuscin-like autofluorescence in fluorescently labeled tissue.
J Histochem Cytochem 47:719–730
60. Shimazaki M, Nakano H, Kobayashi K (2005) Correlation
between tau phosphorylation sites and tangle morphology in
Alzheimer’s disease. Psychogeriatrics 5:22–35
61. Shimura H, Schwartz D, Gygi SP, Kosik KS (2004) CHIP-Hsc70
complex ubiquitinates phosphorylated tau and enhances cell
survival. J Biol Chem 279:4869–4876
62. Sledz P, Zheng H, Murzyn K et al (2010) New surface contacts
formed upon reductive lysine methylation: improving the prob-
ability of protein crystallization. Protein Sci 19:1395–1404
63. Tremblay MA, Acker CM, Davies P (2010) Tau phosphorylated
at tyrosine 394 is found in Alzheimer’s disease tangles and can be
a product of the Abl-related kinase, Arg. J Alzheimers Dis
19:721–733
64. von Bergen M, Friedhoff P, Biernat J, Heberle J, Mandelkow EM,
Mandelkow E (2000) Assembly of tau protein into Alzheimer
paired helical filaments depends on a local sequence motif
((306)VQIVYK(311)) forming beta structure. Proc Natl Acad Sci
USA 97:5129–5134
65. Yuzwa SA, Vocadlo DJ (2009) O-GlcNAc modification and the
tauopathies: insights from chemical biology. Curr Alzheimer Res
6:451–454
66. Zhang B, VerBerkmoes NC, Langston MA, Uberbacher E, Het-
tich RL, Samatova NF (2006) Detecting differential and
correlated protein expression in label-free shotgun proteomics.
J Proteome Res 5:2909–2918
Acta Neuropathol (2012) 123:105–117117
123