(Received for publication, November 7, 1996, and in revised form, February 27, 1997)
From the Neurofilament (NF) proteins are intermediate
filaments found in the neuronal cytoskeleton. Phosphorylation of these
proteins is considered an important factor in the assembly of filaments and determination of filament caliber and stability. Mammalian neurofilaments are composed of three polypeptide subunits, NF-L, NF-M,
and NF-H, all of which are phosphorylated. Here we used techniques for
the mass spectrometric sequencing of proteins from polyacrylamide gels
to analyze in vivo phosphorylation sites on NF-M and NF-L.
Neurofilaments were isolated from rat brain and enzymatically digested
in gel. The resulting peptides were analyzed and sequence data obtained
by nanoelectrospray mass spectrometry. Four phosphorylation sites have
been found in the C-terminal domain of NF-M: serines 603, 608, 666, and
766. Two of these are found in lysine-serine-proline (KSP) motifs and
two in the variant motifs, glutamic acid-serine-proline (ESP) and
valine-serine-proline (VSP). Serine 55 in NF-L was not found to be
phosphorylated, which confirms the possible role of phosphorylation and
dephosphorylation of this site in early neurofilament assembly. The
techniques used enable sequence data and characterization of
posttranslational modifications to be obtained for each individual
subunit directly from polyacrylamide gels.
Neurofilament (NF) proteins, a class of neuronal intermediate
filament proteins, form the principal component of the axonal cytoskeleton. They are composed of three polypeptide subunits, NF-L (61 kDa), NF-M (95 kDa), and NF-H (115 kDa) (1-3), with apparent molecular
masses of 68, 150, and 200 kDa on SDS-polyacrylamide gel
electrophoresis, respectively. They share several structural features
in common with all intermediate filament proteins: a highly conserved
NFPs,1 especially NF-M and NF-H, are known
to be highly phosphorylated in vivo (10-12). Most of the
phosphate is associated with serine residues in KSP motifs within the
C-terminal domains of the larger subunits (10, 11, 13-15). The
phosphorylated tails of these proteins are thought to protrude as side
arms from the assembled filament, enabling cross-linking between
filaments and interactions with other cytoskeletal proteins. These
interactions have been shown to be regulated by phosphorylation
(16-19). Phosphorylation of the tail domains is also thought to
increase spacing between filaments, hence regulating axonal caliber
(20-24) and, in addition, possibly playing a role in the control of
axonal transport (24).
Neurofilaments are also phosphorylated on their N-terminal head domains
(25, 26). This region of NF-L is known to be important in filament
assembly (6, 27, 28), and phosphorylation of intermediate filaments at
their head domains is a proven mechanism for the control of assembly
(29). Serine 55 on NF-L has been found to be phosphorylated in
vivo, yet it displays rapid turnover soon after NF-L synthesis
(30). These findings have led to the suggestion that this site may be
involved to some extent in the regulation of filament assembly and
architecture (12, 30-32).
Abnormal phosphorylation of neurofilaments is associated with some
neurodegenerative diseases such as motor neuron disease (amyotrophic
lateral sclerosis) and the Lewy bodies characteristic of Parkinson's
disease and Lewy body dementia (33-35). Accumulations of highly
phosphorylated neurofilaments, normally found only in axons, are
observed in perikarya and proximal axons in these conditions. This
suggests that aberrant phosphorylation may play a role in the pathology
of these diseases.
To understand the function and importance of neurofilaments as
components of the neuronal cytoskeleton and their involvement in
neurodegenerative disease, it is essential to comprehend the mechanisms
by which these proteins are phosphorylated. Determination of the
endogenous phosphorylation sites is therefore important as this
information will lead to identification of the kinases involved.
Previously, the analysis of sites of NFP phosphorylation has relied
largely on conventional Edman sequencing following lengthy chromatographic separations or two-dimensional phosphopeptide mapping
of proteins radiolabeled with phosphate. Comparison of two-dimensional
peptide maps of NF-L from optic axons of mice intravitreally injected
with 32P with those from in vitro labeled NF-L
has been used to characterize serine 55 as an in vivo
phosphorylation site on NF-L (30). This involved HPLC purification and
subsequent Edman degradation of a phosphopeptide common to both samples
followed by in vitro phosphorylation and sequencing of a
corresponding synthetic peptide to locate the exact site of
phosphorylation. Serine 473 has also been shown to be an in
vivo phosphorylation site on NF-L (36) by Edman sequencing of
HPLC-purified peptides from rat spinal cord corresponding to
phosphopeptides labeled by in vitro phosphorylation. Six
sites on the C-terminal domain of NF-M (37) have been identified by comparison of NF-M peptides from in vitro
32P-labeled cytoskeletal preparations with those from
metabolically labeled dorsal root ganglia. Peptides from unlabeled NF-M
eluting at the same retention time as the labeled phosphopeptides were then sequenced by Edman degradation, and the phosphorylation sites were
determined. Elhanany et al. (38) used a combination of microsequencing and mass spectrometry to identify nine endogenous phosphorylated KSP sites in the C-terminal region of rat NF-H.
Recently, a technique for the sequencing of proteins directly from
polyacrylamide gels using electrospray (39) in combination with tandem
mass spectrometry (40) has been developed (41). This allows sequence
data and characterization of posttranslational modifications, using
parent ion scans (42, 43), to be obtained from total digest mixtures
using, exclusively, mass spectrometric techniques. Proteins are able to
be uniquely identified with the use of peptide tags and data base
searching (44).
We have used this technology in conjunction with small scale
immobilized metal affinity chromatography (IMAC) (45, 46) to identify
phosphopeptides in digest mixtures of NFPs and then to characterize the
sites of phosphorylation. Having covered more than 80% of the sequence
of NF-L, no endogenous phosphorylation sites were found, suggesting
dephosphorylation of the head terminal domain during filament assembly.
Four endogenous phosphorylation sites have been found within the
C-terminal domain of NF-M, including one that has not been previously
reported.
Except where otherwise noted, all chemicals used
were purchased from Sigma and were of the highest quality available.
Hydroxylapatite (HTP) was from Bio-Rad Laboratories Ltd.
(Hertfordshire, UK). Acetic acid and formic acid (AnalaR grade) were
from BDH Laboratory Supplies (Merck Ltd., Leicestershire, UK) The
proteolytic enzymes trypsin (bovine, sequencing grade), endoproteinase
Asp-N (from Pseudomonas fragi, sequencing grade), and
endoproteinase Glu-C (from Staphylococcus aureus strain V8,
sequencing grade) were obtained from Boehringer Mannheim UK Ltd. (East
Sussex, UK). For mass spectrometric analysis and gel spot preparation,
HPLC grade methanol and acetonitrile (Rathburn Chemicals, Scotland)
were used.
A Triton-X-insoluble pellet
was prepared from about 15 g of rat brain by homogenization in
phosphate-buffered saline, pH 7.2, containing KCl (600 mM),
EDTA (2 mM), EGTA (2 mM), phenylmethylsulfonyl fluoride (1 mM), tetrasodium pyrophosphate (10 mM), sodium fluoride (20 mM), sodium
orthovanadate (100 mM), and 0.1% (v/v) Triton X-100, at a
ratio of 1:20 (w/v). After centrifugation (Sorval SS34 rotor, 15,000 rpm, 20 min, 4 °C), the pellet was washed by resuspension in the
same buffer and then in the absence of Triton X-100. The final pellet
was dissolved in buffer A (10 mM sodium phosphate, pH 7.4, made up in 8 M urea containing 1 mM
dithiothreitol), and batchwise HTP fractionation was performed as
described (47). The eluate was dialyzed against buffer B (4 M urea, 20 mM Tris, pH 7.5, 1 mM
dithiothreitol), centrifuged (Beckman type 65 rotor, 50 krpm, 1 h,
15 °C), and loaded on to a Mono Q HR 5/5 column (Pharmacia Biotech
Europe, Sweden). The sample was concentrated by eluting with a gradient
of 0-100% 1 M NaCl in buffer B developed over 10 ml at a
flow rate of 0.5 ml/min.
Approximately 50 µg of NFPs were
separated by one-dimensional SDS-polyacrylamide gel electrophoresis
using precast 8% acrylamide Novex (San Diego, CA) mini-gels (48). Gels
were stained with 0.2% (w/v) Coomassie Brilliant Blue R250 in 50%
(v/v) methanol in water containing 2% (v/v) acetic acid and destained
in 50% (v/v) methanol, 2% (v/v) acetic acid. Protein bands were cut
from the gel, reduced, acetamidated, and tryptically digested as
described (41). Peptides were extracted twice with 100 mM
NH4HCO3 and acetonitrile followed by two
extractions with 5% (v/v) acetic acid and acetonitrile and then dried.
Additional gels were run in which samples were also subjected to
enzymatic digestion using endoproteinase Glu-C (50 mM
NH4HCO3, pH 7.8, 25 °C) and endoproteinase Asp-N (50 mM sodium phosphate buffer, pH 8.0, 37 °C) by
the same method.
Empty miniature
Protein Chemistry Systems (PCS) desalting columns (Hewlett-Packard,
Cheshire, UK) were packed with chelating Sepharose high performance
slurry (70 µl in 20% (v/v) ethanol, Pharmacia) and washed with water
(2 ml) followed by 0.1 M acetic acid, pH 3.1 (solution A,
500 µl). 0.1 M FeCl3 solution (50 µl in
solution A) was applied followed by washing with solution A (500 µl)
to remove excess iron. The dried peptide mixture in the gel digest was
then loaded (dissolved in 50 µl of solution A), and the column was
washed with buffer A (500 µl). The phosphopeptides were eluted with
0.1 M Tris-HCl (300 µl), pH 8.5, and the eluate dried
before being analyzed by nanoelectrospray mass spectrometry.
Needles
for nanoelectrospray mass spectrometry were made with a micropipette
puller (Sutter Instrument Co., Novato, CA) from borosilicate glass
capillaries (Clark Electromedical Instruments, Pangbourne, Reading, UK)
as described by Wilm and Mann (49). They were gold-coated in a vapor
desorption instrument. Dried protein digests were dissolved in 5%
(v/v) formic acid and desalted on a miniature PCS column self-packed
with ~20 µl of POROS R2 sorbent (PerSeptive Biosystems, Framingham,
MA) as described (41). The sample was not eluted directly into the
spraying needle but was dried and then taken up in 10 µl of spraying
solution (50% (v/v) methanol, 1% (v/v) formic acid in water for
positive ion or 50% (v/v) methanol, 5% (v/v) ammonia in water for
negative ion), and 1 µl was inserted into the needle. Electrospray
mass spectra were acquired on an API III triple quadrupole machine (Perkin-Elmer Sciex, Ontario, Canada) equipped with a nanoES ion source
developed by Wilm and Mann (49, 50). Q1 scans were performed with 0.1-Da mass step. For operation in the MS/MS mode, Q1 was set to transmit a mass window of 2 Da for both
parent and product ion scans, and spectra were accumulated with 0.2-Da
mass steps. Dwell time was 1 ms for all scans except for parent ion scans, where it was 3 ms. Resolution was set so that fragment masses
could be assigned to more than 1 Da. Collision energy was individually
tuned for each peptide for optimum MS/MS spectra. A new needle was used
for each experiment. Spectra interpretation was performed using
BioMultiView (Sciex) software.
NFPs purified from rat brain were
run on SDS-polyacrylamide gel electrophoresis, and NF-M and NF-L were
tryptically digested in gel. After desalting on POROS, analysis of the
total digest mixtures of NF-M and NF-L was performed by nanoES mass
spectrometry. Fragmentation of peptide ions by collision-induced
dissociation (CID) tandem MS resulted in partial sequences being
obtained that, in combination with the mass, were sufficient to
characterize unambiguously the peptides from the known protein
sequences (1, 2, 44).
Phosphopeptides were isolated from the digest mixture using a small
scale IMAC technique, which takes advantage of the affinity of
phosphopeptides for immobilized Fe3+ ions (45). Following
small scale desalting, the samples were analyzed by nanoES MS/MS.
Phosphopeptides were identified within the digest mixtures using scans
for the parents of m/z 79 in negative ion mode
(42, 43) and, for neutral loss of 49, [M-H3PO4 + 2H]2+, in positive
ion mode (53). The parent ion scan shows ions that fragment to produce
an ion at m/z 79 (the phospho group, PO3 The Q1 spectra of IMAC-purified samples mainly showed only
ions corresponding to phosphopeptides in comparison with the total digest spectra (Fig. 1, A and B).
A few non-phosphopeptides containing histidine residues were also seen,
since histidine is thought to have some affinity to the packing used.
The use of the IMAC column reduced the problem of electrospray ion
suppression by reduction of the complexity of the peptide mixture. This
reduction increased the phosphorylated peptide ion signal strength and
thus improved the quality of the product ion spectra.
Mapping phosphopeptides in NF-M using
IMAC. NF-M was tryptically digested in gel, and the peptides were
analyzed by nanoES MS. Scans for ions indicative of phosphopeptides
were used to identify those present: P1,
AKpSPVPKpSPVEEVKPKPEAK; P2,
AKSPVPKpSPVEEVKPKPEAK; P3, SPVPKpSPVEEVKPKPEAK; P4, GVVTNGLDVpSPAEEK; P5,
GSGQEEEKGVVTNGLDVpSPAEEK; P6, KAEpSPVKEK.
A, positive ion Q1 mass spectrum of the tryptic
peptide mixture. B, positive ion Q1 mass
spectrum of IMAC-separated sample. C, parent ion scan for
the phospho group in negative ion mode. This shows the massses of ions
that fragment to produce an ion at m/z 79 (PO3
Four phosphorylation sites, which must have been
generated in vivo, were found within the C-terminal region
of NF-M. Three of these, two in KSP motifs and one in the variant ESP
motif, have been reported previously by Xu et al. (37) on
the basis of metabolically labeled dorsal root ganglia. The fourth site has not been reported before in NF-M and lies within a VSP motif.
Fig. 1 shows the identification of phosphopeptides in the NF-M tryptic
digest. The total digest mixture was simplified by the use of IMAC
(Fig. 1, A and B), and the resulting
Q1 scan revealed several potential phosphopeptides (Fig.
1B). The parents of m/z 79 scan in
negative ion mode was then used to further narrow the field of
candidate phosphopeptides (Fig. 1C) to five. The neutral loss of 49 scan in positive ion mode (Fig. 1D) revealed an
additional phosphopeptide and confirmed the presence of those
identified in the parent ion scan.
The peptides and sites of phosphorylation were then identified by CID
tandem mass spectrometry in positive ion mode, resulting in product ion
spectra (Fig. 2). Sequence tags were constructed from
the resulting fragment ion masses, and hence peptides were identified
from the published sequence (Table I, Ref. 2).
Table I.
Phosphopeptides identified in the enzymatic digests of NF-M
Department of Neuroscience,
-helical rod domain, essential for filament formation (4, 5), a
short N-terminal head that may be involved in the regulation of
filament assembly (6), and a C-terminal domain of variable length
(7-9).
Materials
Analysis of Tryptic Digests
). The neutral loss scan shows the
masses of precursor ions that lose the phosphate group as a neutral
fragment.
Fig. 1.
). D, scan for neutral
loss of the phosphate group in positive ion mode. This shows the masses of precursor ions that lose the phosphate group as a neutral fragment,
[M-H3PO4 + 2H]2+.
[View Larger Version of this Image (19K GIF file)]
Fig. 2.
Tandem mass spectrometry of NF-M
phosphopeptides. A, positive ion tandem mass spectrum of
m/z 769.2, identifying the peptide and sites of
phosphorylation as AKpSPVPKpSPVEEVKPKPEAK (P1).
B, positive ion tandem mass spectrum of
m/z 798.2, identifying the phosphopeptide as
GVVTNGLDVpSPAEEK (P4). The phosphoserine residue can be
located as indicated.
[View Larger Version of this Image (26K GIF file)]
Phosphopeptide
Residue no.
Peptide sequence
P1
601-620
(K)AKpSPVPKpSPVEEVKPKPEAK(A)
P2
601-620
(K)AKSPVPKpSPVEEVKPKPEAK(A)
P3
603-620
(K)SPVPKpSPVEEVKPKPEAK(A)
P4
757-771
(K)GVVTNGLDVpSPAEEK(K)
P5
749-771
(K)GSGQEEEKGVVTNGLDVpSPAEEK(K)
P6
663-671
(K)KAEpSPVKEK(A)
P7
600-612
(E)KAKSPVPKpSPVEE(V)
P8
593-612 or
(E)IKVEKPEKAKpSPVPKpSPVEE(V) or
592-611
(E)EIKVEKPEKAKpSPVPKpSPVE(E)
P9
597-618
(E)KPEKAKSPVPKpSPVEEVKPKPE(A) or
(E)KPEKAKpSPVPKSPVEEVKPKPE(A)
Fragmentation of the 3+ ion of P1 (m/z 769.6) in positive ion mode gave the partial sequence 603pSPVP from doubly charged Y" ions (Fig. 2A), which, from the known sequence, is sufficient to characterize the peptide and sites of phosphorylation as residues 601-620 with both serines phosphorylated (AK603pSPVPK608pSPVEEVKPKPEAK, Mr 2305.3). Fragments corresponding to the loss of 608pS were also seen as singly charged b ions.
MS/MS of the 3+ ion (m/z 742.1) of P2 resulted in doubly charged Y" ions, giving the partial sequence SPVPK608pS, which confirmed the identity of the peptide as residues 601-620 with serine 608 phosphorylated (Mr 2225.4). This indicates that serine 603 is heterogeneously phosphorylated within the NF-M molecule.
The product ion spectrum of the 3+ ion of P3 (m/z 676.0) displayed doubly charged Y" ion fragments corresponding to the sequence VPK and confirming the identity of the peptide as residues 603-620 with serine 608 phosphorylated (Mr 2026.2). This is the expected site for phosphorylation on this peptide since trypsin has cleaved at lysine 602, which it may not have done had serine 603 been phosphorylated.
Fragmentation of the 2+ ion of P4 (m/z 798.2) gave a full series of Y" ions (Fig. 2B), thereby characterizing the peptide as residues 757-771 and confirming that serine 766 is phosphorylated (GVVTNGLDV766pSPAEEK, Mr 1594.6). The unphosphorylated form of this peptide was also characterized, again indicating heterogeneity of phosphorylation. The signal-to-noise ratio of the CID spectrum of P5 was not sufficient to produce any useful sequence information.
CID MS/MS of the ion at m/z 546.4 corresponding to the 2+ of P6 gave a series of b ions (sequence VK) that enabled characterization of the peptide as KAE666pSPVKEK (residues 663-671, Mr 1095.1.) with serine 666 carrying a phosphate.
Analysis of Endoproteinase Glu-C and Asp-N DigestsNF-M and
NF-L were digested in gel with endoproteinases Glu-C and Asp-N.
Desalted samples were analyzed by nanoES MS/MS, and the peptides were
identified, thus giving greater sequence coverage for each protein.
Total sequence coverage gained for NF-L was 81% and for NF-M, 64%
(Fig. 3).
Endoproteinase Glu-C-digested samples were subjected to IMAC and then analyzed by nanoES MS/MS. Scans for fragment masses characteristic of phosphopeptides were performed. The negative ion parents of m/z 79 scan (not shown) revealed two phosphopeptides in the NF-M sample corresponding to phosphorylation at serines 603 and 608 (P8 and P9, Table I). The signal-to-noise ratio of the CID spectra of these peptides was not sufficient to produce any useful sequence information. Fragmentation in positive ion mode of the ion at m/z 738.4 seen in the neutral loss scan (not shown) gave sequence data corresponding to residues 600-612, with serine 608 phosphorylated (P7, Table I).
Analysis of NF-L81% of the sequence of NF-L was covered by mass spectrometric sequencing; however, no phosphopeptides were found within the NF-L molecule. IMAC of the digest mixtures also failed to reveal any phosphopeptides. Upon phosphorylation with cAMP-dependent protein kinase, phosphorylated residues were identified within the NF-L molecule using this technique,2 which suggests that the absence of endogenous sites was not due to a technical problem. These findings seem consistent with the notion that NF-L is mainly phosphorylated at its head domain and that these phosphates are removed before assembly into filaments. The glutamic acid-rich tail region of NF-L containing 74 residues, including serine 473, which has been found to be phosphorylated in vivo (36), could not be located despite the use of several different proteases. This may perhaps be due to incomplete digestion of this region or suppression of ionization of the peptides in the total digest mixture by these residues.
Phosphorylation of neurofilament proteins is considered an important factor in the assembly of filaments, determination of filament caliber, and stability and plays a potential role in the pathology of several neurodegenerative diseases. Until now, conventional approaches have been employed in the analysis of phosphorylation of NFPs. These methods have involved metabolic labeling, either in vivo or of cultured neurons, and two-dimensional phosphopeptide mapping or lengthy chromatographic separations after various enzymatic digestions. Phosphopeptides have then been sequenced and sites located by the use of conventional protein sequencing. These methods nearly always require the purification to homogeneity of often limited quantities of the neurofilament subunits and separation of the digest mixtures and are limited by the sensitivity of Edman degradation.
Here we use techniques developed by Mann and co-workers (41) for the mass spectrometric sequencing of proteins directly from polyacrylamide gels, hence eleviating the necessity for subunit separation and enabling sequencing of peptides from a total digest mixture at lower sensitivity levels. The nature of the fragments generated by tandem MS of peptides allows unique characterization of the peptide, both from an unknown protein with the use of peptide tags and data base searching (44) and, as in this case, from a known protein sequence.
Phosphopeptides were identified within the digest mixtures by the use of parent ion (42, 43) and neutral loss (53) scans. Once distinguished from the peptide mixture, the phosphopeptides could be fragmented during the same experiment, yielding sequence data and hence uniquely characterizing the peptide and site of phosphorylation. The extremely low flow rate of the nanospray technique (49) requires the use of just 1 µl of sample/30 min of analysis and therefore enables the acquisition of a large quantity of sequence data with low sample consumption.
A small scale IMAC technique was developed using mini-desalting columns, supplied empty (Hewlett Packard), which were self-packed with a small amount of chelating Sepharose high performance slurry (Pharmacia). This enabled small scale separation of phosphopeptides from the digest mixture due to their affinity for Fe3+ ions (45), resulting in improved sensitivity for the phosphopeptide ions and augmenting the quality of the sequence data obtained. This can be explained due to the fact that in a mixture of peptides, the more strongly ionizing components can often suppress the signal from those that are more weakly ionizing. Therefore, by reducing the complexity of the mixture by IMAC, the likelihood of suppression is also reduced (46).
Using these methods, we have characterized four phosphorylation sites within the C-terminal domain of NF-M and covered 64% of the sequence by mass spectrometric sequencing (Fig. 3). Three of the sites have been reported previously; two in KSP motifs and one in the variant motif, ESP. In addition, we have characterized a novel site found within a VSP motif that may also be phosphorylated by a proline-directed kinase.
We have also been able to gain some insight in the heterogeneity of phosphorylation of some of these residues. Serine 603 has been found in both its phosphorylated and non-phosphorylated forms, as has serine 766. There is evidence to suggest that the phosphorylation of NFPs is heterogeneous (54, 55) and that the state of phosphorylation changes as the proteins are transported down the axon (56). Our results are in agreement with these findings and identify particular sites displaying this heterogeneity, although we cannot unambiguously rule out loss of phosphate during neurofilament manipulation.
We have covered 81% of the NF-L sequence (Fig. 3) by mass spectrometric sequencing but found no sites of phosphorylation. The fact that no sites were found in the head region of NF-L seems to confirm the hypothesis that these sites undergo phosphorylation and dephosphorylation during filament assembly (12, 30, 31), since we started with assembled neurofilaments for the preparation. When we phosphorylated NF-L with cAMP-dependent protein kinase, which is known to phosphorylate the head domain of NF-L (30, 57), we were able to identify sites of phosphorylation by mass spectrometric sequencing,2 which suggests that the lack of endogenous sites found is a true result rather than a practical limitation.
Some regions of the sequence of both proteins, namely those that are rich in glutamic acid residues, remain uncovered. This may be caused either by incomplete digestion or by poor ionization of these peptides due to the presence of these residues.
In addition to phosphorylation, neurofilaments have been shown to be posttranslationally modified by O-linked N-acetylglucosamine (51, 52). The parent ion scan approach may also be used in the identification of glycopeptides in a digest mixture (42) as N-hexosamines give a characteristic oxonium ion at m/z 204. We have used this scan for parents of m/z 204 in the analysis of NF-M and NF-L but found no evidence of glycopeptides. This may be due to the relatively low amounts of these species within neurofilaments (51, 52) compared with those modified by phosphorylation (11).
The sensitivity of this technique, coupled with the ease and speed of sample preparation and analysis, should enable NFPs and other proteins to be analyzed for posttranslational modifications from a variety of sources. These may include cells stimulated to activate signal transduction cascades and tissue from diseases such as motor neuron disease.