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2002
Microcolumns with self-assembled particle frits
for proteomics
J Chromatogr A. 2002 Dec
6;979(1-2):233-9.
Yasushi Ishihama, Juri Rappsilber, Jens
S. Andersen and Matthias Mann
LC–MS–MS experiments in
proteomics are usually performed with packed microcolumns employing frits
or outlets smaller than the particle diameter to retain the packing
material. We have developed packed microcolumns using self-assembled
particles (SAPs) as frits that are smaller than the size of the outlet. A
five to one ratio of outlet size to particle diameter appears to be the
upper maximum. In these situations the particles assembled into an arch
over the outlet like the stones in a stone bridge. When 3 um particles were
packed into a tapered column with an 8 um outlet, two particles bridged the
outlet with 0.3 pl dead volume and perfect success rate. In peptide
analysis by LC–MS, the peak width at half height was normally less
than 6 s, compared to 12 s without SAPs. The LC–MS–MS system
provided 37% sequence coverage (21 matched peptides) for a
tryptically-digested sample of 10 fmol bovine serum albumin. We also
describe application of the SAP principle to make disposable pipette tip
columns with short pieces of fused-silica capillary as the outlet.
Analysis
of Tyrosine Phosphorylation Sites in Signaling Molecules by a
Phosphotyrosine-Specific Immonium Ion Scanning Method
Science's STKE:
http://stke.sciencemag.org/cgi/content/full/sigtrans;2002/154/pl16
[Abstract]
[Full
Text]
Hanno Steen, Akhilesh Pandey, Jens S.
Andersen, and Matthias Mann (15 Oct 2002)
Signal transduction pathways involve
cascades of events, such as formation of second messengers and protein
complexes that alter the activities of proteins. This can ultimately lead
to changes in gene expression in response to the stimuli. Reversible
phosphorylation of proteins is an important mechanism for activating or
inhibiting enzymes and for the assembly of multiprotein complexes. Here, we
describe a mass spectrometry-based phosphotyrosine-specific immonium ion
scanning (PSI scanning) method for selective detection of
tyrosine-phosphorylated peptides. Once the tyrosine-phosphorylated peptides
are identified, they can be directly sequenced in the same experiment to
localize the phosphorylation site. We provide protocols for isolation and
preparation of samples for analysis, and detailed instructions for
operation of a quadrupole time-of-flight (TOF) mass spectrometer for this
method. Because of its simplicity and specificity, PSI scanning is likely
to become an important tool in proteomic studies of pathways involving
tyrosine phosphorylation.
Analysis
of the Plasmodium falciparum proteome by high-accuracy mass
spectrometry
Nature 419, 537 - 542 (2002)
EDWIN LASONDER,
YASUSHI ISHIHAMA, JENS S. ANDERSEN,
ADRIAAN M. W. VERMUNT, ARNAB PAIN,
ROBERT W. SAUERWEIN, WIJNAND M. C. ELING,
NEIL HALL, ANDREW P. WATERS, HENDRIK G. STUNNENBERG
& MATTHIAS MANN
The annotated genomes of organisms
define a 'blueprint' of their possible gene products. Post-genome analyses
attempt to confirm and modify the annotation and impose a sense of the
spatial, temporal and developmental usage of genetic information by the
organism. Here we describe a large-scale, high-accuracy (average deviation
less than 0.02 Da at 1,000 Da) mass spectrometric proteome
analysis of selected stages of the human malaria parasite Plasmodium
falciparum. The analysis revealed 1,289 proteins of which 714 proteins were
identified in asexual blood stages, 931 in gametocytes and 645 in gametes.
The last two groups provide insights into the biology of the sexual stages
of the parasite, and include conserved, stage-specific, secreted and
membrane-associated proteins. A subset of these proteins contain domains
that indicate a role in cell–cell interactions, and therefore can be
evaluated as potential components of a malaria vaccine formulation. We also
report a set of peptides with significant matches in the parasite genome but
not in the protein set predicted by computational methods.
RGM is a repulsive guidance molecule for retinal
axons.
Nature 2002 Sep 26;419(6905):392-5
Monnier PP, Sierra A, Macchi P,
Deitinghoff L, Andersen JS, Mann M, Flad M, Hornberger MR, Stahl B,
Bonhoeffer F, Mueller BK.
Axons rely on guidance cues to reach
remote targets during nervous system development. A well-studied model
system for axon guidance is the retinotectal projection. The retina can be
divided into halves; the nasal half, next to the nose, and the temporal
half. A subset of retinal axons, those from the temporal half, is guided by
repulsive cues expressed in a graded fashion in the optic tectum, part of
the midbrain. Here we report the cloning and functional characterization of
a membrane-associated glycoprotein, which we call RGM (repulsive guidance
molecule). This molecule shares no sequence homology with known guidance
cues, and its messenger RNA is distributed in a gradient with increasing
concentration from the anterior to posterior pole of the embryonic tectum.
Recombinant RGM at low nanomolar concentration induces collapse of temporal
but not of nasal growth cones and guides temporal retinal axons in vitro,
demonstrating its repulsive and axon-specific guiding activity.
Inhibition
of adipocyte differentiation by Resistin like molecule alpha (RELM alpha):
Biochemical characterization of its oligomeric nature.
J Biol Chem 2002 Aug 19; [epub ahead of print]
Blagoev B, Kratchmarova I, Nielsen
MM, Fernandez MM, Voldby J, Andersen JS, Kristiansen K, Pandey A,
Mann M.
A novel family of cysteine-rich secreted proteins with unique tissue
distribution has recently been identified. One of the members, resistin
(for resistance to insulin), also called FIZZ3, was identified in a screen
for molecules that are downregulated in mature adipocytes upon
administration of thiazolidinediones. The prototypical member of this
family was originally identified from bronchoalveolar lavage fluid of
inflamed lungs and designated FIZZ1 (found in inflammatory zone). This
molecule was also found to be highly expressed in adipose tissue and was
named resistin like molecule a (RELMa). Here we demonstrate that RELMa
inhibits the differentiation of 3T3-L1 preadipocytes into adipocytes. RELMa
has no effect on proliferation of 3T3-L1 preadipocytes. Pretreatment of
3T3-L1 preadipocytes with RELMa does not affect insulin or PDGF induced
mitogenesis. IRS-1 phosphorylation and glucose transport stimulated by
insulin in mature adipocytes were also unaffected by RELMa. We show that
RELMa forms disulfide-linked homooligomers based on results from
electrophoresis under reducing and non-reducing conditions,
coimmunoprecipitation experiments as well as by mass spectrometry. In
addition, RELMa is able to form heterooligomers with resistin but not
RELMb. Since RELMa is expressed by adipose tissue and it is a secreted
factor, our finding suggest that RELMa may be involved in the control of
the adipogenesis as well as in the process of muscle differentiation.
Large-Scale Proteomic Analysis of the Human
Spliceosome,
Genome Res. Jul 22
(2002). [epub ahead of
print].
Rappsilber, J.,Ryder, U., Lamond, A. I., and Mann, M.
In a previous proteomic study of the human spliceosome, we identified 42
spliceosome-associated factors, including 19 novel ones. Using enhanced mass spectrometric tools and
improved databases, we now report
identification of 311 proteins that copurify with splicing complexes assembled on two separate
pre-mRNAs. All known essential human
splicing factors were found, and 96 novel proteins were identified, of which 55 contain domains
directly linking
them to functions in
splicing/RNA processing. We also detected 20 proteins related to transcription, which
indicates a direct connection between
this process and splicing. This investigation provides the most detailed inventory of human
spliceosome-associated factors to date, and
the data indicate a number of interesting links coordinating splicing with other steps in
the gene expression pathway.
Supplementary Material (PIL)
Stable
isotope labeling by amino acids in cell culture, SILAC, as a simple and
accurate approach to expression proteomics
Mol Cell Proteomics. 2002 May;1(5):376-86.
Shao-En Ong, Blagoy Blagoev, Irina
Kratchmarova, Dan Bach Kristensen, Hanno Steen, Akhilesh
Pandey, and Matthias Mann.
Quantitative
proteomics has traditionally been performed by 2D gel electrophoresis but
recently, mass spectrometric methods based on stable isotope quantitation
have shown great promise for the simultaneous and automated identification
and quantitation of complex protein mixtures. Here we describe a method,
termed SILAC for Stable Isotope Labeling by Amino acids in Cell culture,
for the in vivo incorporation of specific amino acids into all mammalian
proteins. Mammalian cell lines are grown in media lacking a standard
essential amino acid but supplemented with a non-radioactive, isotopically
labeled form of that amino acid, in this case deuterated leucine (Leu-d3).
We find that growth of cells maintained in these media is no different from
growth in normal media as evidenced by cell morphology, doubling time and
ability to differentiate. Complete incorporation of Leu-d3 occurred after
five doublings in the cell lines and proteins studied. Protein populations
from experimental and control samples are mixed directly after harvesting
and mass spectrometric identification is straightforward as every
leucine-containing peptide incorporates either all normal leucine or all
Leu-d3. We have applied this technique to the relative quantitation of
changes in protein expression during the process of muscle cell
differentiation. Proteins that were found to be upregulated during this
process include glyceraldehyde-3-phosphate dehydrogenase, fibronectin and
pyruvate kinase M2. SILAC is a simple, inexpensive and accurate procedure
that can be used as a quantitative proteomic approach in any cell culture
system.
A
Proteomic Approach for Identification of Secreted Proteins during the
Differentiation of 3T3-L1 Preadipocytes to Adipocytes.
Mol Cell Proteomics 2002 Mar;1(3):213-22
Kratchmarova
I, Kalume DE, Blagoev B, Scherer PE, Podtelejnikov AV, Molina H, Bickel PE,
Andersen JS, Fernandez MM, Bunkenborg J, Roepstorff P, Kristiansen K,
Lodish HF, Mann M, Pandey A
We have
undertaken a systematic proteomic approach to purify and identify secreted
factors that are differentially expressed in preadipocytes versus
adipocytes. Using one-dimensional gel electrophoresis combined with
nanoelectrospray tandem mass spectrometry, proteins that were specifically
secreted by 3T3-L1 preadipocytes or adipocytes were identified. In addition
to a number of previously reported molecules that are up- or down-regulated
during this differentiation process (adipsin, adipocyte complement-related
protein 30 kDa, complement C3, and fibronectin), we identified four
secreted molecules that have not been shown previously to be expressed
differentially during the process of adipogenesis. Pigment
epithelium-derived factor, a soluble molecule with potent antiangiogenic
properties, was found to be highly secreted by preadipocytes but not
adipocytes. Conversely, we found hippocampal cholinergic neurostimulating
peptide, neutrophil gelatinase-associated lipocalin, and haptoglobin to be
expressed highly by mature adipocytes. We also used liquid
chromatography-based separation followed by automated tandem mass
spectrometry to identify proteins secreted by mature adipocytes. Several
additional secreted proteins including resistin, secreted acidic
cysteine-rich glycoprotein/osteonectin, stromal cell-derived factor-1,
cystatin C, gelsolin, and matrix metalloprotease-2 were identified by this
method. To our knowledge, this is the first study to identify several novel
secreted proteins by adipocytes by a proteomic approach using mass spectrometry.
Analysis
of protein phosphorylation using mass spectrometry: deciphering the
phosphoproteome.
Trends Biotechnol 2002 Jun;20(6):261-8
Mann M, Ong SE, Gronborg M, Steen H, Jensen ON, Pandey A.
Center for Experimental Bioinformatics, University of Southern
Denmark, M, DK-5230, Odense, Denmark
In signal transduction in eukaryotes, protein phosphorylation is a key
event. To understand signaling processes, we must first acquire an
inventory of phosphoproteins and their phosphorylation sites under
different conditions. Because phosphorylation is a dynamic process,
elucidation of signaling networks also requires quantitation of these
phosphorylation events. In this article, we outline several methods for
enrichment of phosphorylated proteins and peptides and discuss various
options for their identification and quantitation with special emphasis on
mass spectrometry-based techniques.
Axin-mediated
CKI phosphorylation of beta-catenin at Ser 45: a molecular switch for the
Wnt pathway.
Genes Dev 2002 May 1;16(9):1066-76
Amit S,
Hatzubai A, Birman Y, Andersen JS, Ben-Shushan E, Mann M, Ben-Neriah Y,
Alkalay I.
The Wnt pathway controls numerous developmental processes via the
beta-catenin-TCF/LEF transcription complex. Deregulation of the pathway
results in the aberrant accumulation of beta-catenin in the nucleus, often
leading to cancer. Normally, cytoplasmic beta-catenin associates with APC
and axin and is continuously phosphorylated by GSK-3beta, marking it for
proteasomal degradation. Wnt signaling is considered to prevent GSK-3beta
from phosphorylating beta-catenin, thus causing its stabilization. However,
the Wnt mechanism of action has not been resolved. Here we study the
regulation of beta-catenin phosphorylation and degradation by the Wnt
pathway. Using mass spectrometry and phosphopeptide-specific antibodies, we
show that a complex of axin and casein kinase I (CKI) induces beta-catenin
phosphorylation at a single site: serine 45 (S45). Immunopurified axin and
recombinant CKI phosphorylate beta-catenin in vitro at S45; CKI inhibition
suppresses this phosphorylation in vivo. CKI phosphorylation creates a
priming site for GSK-3beta and is both necessary and sufficient to initiate
the beta-catenin phosphorylation-degradation cascade. Wnt3A signaling and
Dvl overexpression suppress S45 phosphorylation, thereby precluding the
initiation of the cascade. Thus, a single, CKI-dependent phosphorylation
event serves as a molecular switch for the Wnt pathway.
What does it mean to identify a protein in proteomics?
Trends Biochem Sci 2002 Feb 1;27(2):74-78
Rappsilber J, Mann M.
Protein Interaction Laboratory in the Center of Experimental
Bioinformatics, Dept of Biochemistry and Molecular Biology, University of
Southern Denmark, Campusvej 55, DK-5230 M, Odense, Denmark
The annotation of the human genome indicates the surprisingly low number of
[similar]40000 genes. However, the estimated number of proteins encoded by
these genes is two to three orders of magnitude higher. The ability to
unambiguously identify the proteins is a prerequisite for their functional
investigation. As proteins derived from the same gene can be largely
identical, and might differ only in small but functionally relevant
details, protein identification tools must not only identify a large number
of proteins but also be able to differentiate between close relatives. This
information can be generated by mass spectrometry, an approach that
identifies proteins by partial analysis of their digestion-derived
peptides. Information gleaned from databases fills in the missing sequence
information. Because both sequence databases and experimental data are limited,
a certain ambiguity often remains concerning which sequence variant(s) and
modification(s) are present. As the common denominator of all the isoforms
is a gene, in our opinion, it would be more accurate to state that a
product of this particular gene rather than a certain protein has been
identified by mass spectrometry
A
Novel WD Repeat Protein Component of the Methylosome Binds Sm Proteins.
J Biol Chem 2002 Mar 8;277(10):8243-8247
Friesen WJ, Wyce A, Paushkin S, Abel L, Rappsilber J, Mann M, Dreyfuss
G.
Howard Hughes Medical Institute and Department of Biochemistry and
Biophysics, University of Pennsylvania School of Medicine, Philadelphia,
Pennsylvania 19104-6148 and the Protein Interaction Laboratory, Center for
Experimental Bioinformatics and Department of Biochemistry and Molecular
Biology, University of Southern Denmark, DK-5230 Odense M, Denmark.
We have recently described a large (20 S) protein arginine
methyltransferase complex, termed the methylosome, that contains the
methyltransferase JBP1 (PRMT5) and the pICln protein. The methylosome
functions to modify specific arginines to dimethylarginines in the
arginine- and glycine-rich domains of several spliceosomal Sm proteins, and
this modification targets these proteins to the survival of motor neurons
(SMN) complex for assembly into small nuclear ribonucleoprotein (snRNP)
core particles. Here, we describe a novel component of the methylosome, a
50-kilodalton WD repeat protein termed methylosome protein 50 (MEP50). We
show that MEP50 is important for methylosome activity and binds to JBP1 and
to a subset of Sm proteins. Because WD repeat proteins provide a platform
for multiple protein interactions, MEP50 may function to mediate the
interaction of multiple substrates with the methylosome. Interestingly, all
of the known components of the methylosome bind Sm proteins, suggesting
that in addition to producing properly methylated substrates for the SMN
complex, the methylosome may be involved in Sm protein rearrangements or
pre-assembly required for snRNP biogenesis.
Purification
of Native Survival of Motor Neurons Complexes and Identification of Gemin6
as a Novel Component.
J Biol Chem 2002 Mar 1;277(9):7540-7545
Pellizzoni L, Baccon J, Rappsilber J, Mann M, Dreyfuss G.
Howard Hughes Medical Institute and Department of Biochemistry &
Biophysics, University of Pennsylvania School of Medicine, Philadelphia,
Pennsylvania 19104-6148 and the Protein Interaction Laboratory, Center for
Experimental Bioinformatics and Department of Biochemistry & Molecular
Biology, University of Southern Denmark, DK-5230 Odense M, Denmark.
The survival of motor neurons (SMN) protein, the product of the gene
responsible for the motor neuron degenerative disease spinal muscular
atrophy (SMA), is part of a large macromolecular complex. The SMN complex
is localized in both the cytoplasm and the nucleus and contains SMN, Gemin2,
Gemin3, Gemin4, Gemin5, and a few not yet identified proteins. The SMN
complex plays a key role in the biogenesis of spliceosomal small nuclear
ribonucleoproteins (snRNPs) and other ribonucleoprotein particles. As a
step toward the complete characterization of the components of the SMN
complex, we generated stable cell lines that express FLAG-tagged SMN or
Gemin2 under the control of a tetracycline-inducible promoter. Native SMN
complexes of identical protein composition to those isolated by immunoprecipitation
with anti-SMN antibodies were purified by affinity chromatography from
extracts of both cell lines. Here we report the identification by mass
spectrometry of a novel protein component of the SMN complex termed Gemin6.
Co-immunoprecipitation, immunolocalization, and in vitro binding
experiments demonstrate that Gemin6 is a component of the SMN complex that
localizes to gems and interacts with several Sm proteins of the
spliceosomal snRNPs.
Gemin5,
a novel WD repeat protein component of the SMN complex that binds Sm
proteins.
J Biol Chem 2002 Feb 15;277(7):5631-6
Gubitz AK, Mourelatos Z, Abel L, Rappsilber J, Mann M, Dreyfuss G.
Howard Hughes Medical Institute, Department of Biochemistry, University of
Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6148,
USA.
The survival of motor neurons (SMN) protein is the product of the disease
gene of spinal muscular atrophy and is found both in the cytoplasm and the
nucleus, where it is concentrated in gems. SMN is part of a multi-protein
complex that includes Gemin2, Gemin3, and Gemin4. The SMN complex plays an
important role in the cytoplasmic assembly of small nuclear
ribonucleoproteins (snRNPs) and likely other RNPs in pre-mRNA splicing and
in the assembly of transcriptosomes. Here, we report the identification of
an additional component of the SMN complex, a novel WD repeat protein
termed Gemin5. Gemin5 binds SMN directly and is a component of the SMN
complex. Furthermore, Gemin5 interacts with several of the snRNP core
proteins including SmB, SmD1, SmD2, SmD3, and SmE, suggesting that it
participates in the activities of the SMN complex in snRNP assembly. Immunolocalization
studies demonstrate that Gemin5 is found in the cytoplasm and in the
nucleus, where it colocalizes with SMN in gems. The presence of 13 WD
repeat domains in the amino-terminal half of Gemin5 and a coiled-coil motif
near its carboxyl terminus indicate that it may form a large heteromeric
complex and engage in multiple interactions.
YUEN HO*,
ALBRECHT GRUHLER*, ADRIAN HEILBUT*,
GARY D. BADER†‡, LYNDA MOORE*,
SALLY-LIN ADAMS*, ANNA MILLAR*, PAUL TAYLOR*,
KEIRYN BENNETT*, KELLY BOUTILIER*, LINGYUN YANG*,
CHERYL WOLTING*, IAN DONALDSON*, SגEN SCHANDORFF*,
JUANITA SHEWNARANE*, MAI VO*†, JOANNE TAGGART*†,
MARILYN GOUDREAULT*†, BRENDA MUSKAT*, CRIS ALFARANO*,
DANIELLE DEWAR†, ZHEN LIN†,
KATERINA MICHALICKOVA†‡,
ANDREW R. WILLEMS†§, HOLLY SASSI†, PETER A. NIELSEN*,
KARINA J. RASMUSSEN*, JENS R. ANDERSEN*,
LENE E. JOHANSEN*, LYKKE H. HANSEN*,
HANS JESPERSEN*, ALEXANDRE PODTELEJNIKOV*, EVA NIELSEN*,
JANNE CRAWFORD*, VIBEKE POULSEN*,
BIRGITTE D. SגENSEN*, JESPER MATTHIESEN*,
RONALD C. HENDRICKSON, FRANK GLEESON, TONY PAWSON,
MICHAEL F. MORAN, DANIEL DUROCHER, MATTHIAS MANN,
CHRISTOPHER W. V. HOGUE, DANIEL FIGEYS &
MIKE TYERS. Systematic
identification of protein complexes in Saccharomyces cerevisiae by
mass spectrometry Nature
415, 180 - 183 (2002)
The recent abundance of genome sequence
data has brought an urgent need for systematic proteomics to decipher the
encoded protein networks that dictate cellular function. To date,
generation of large-scale protein–protein interaction maps has relied
on the yeast two-hybrid system, which detects binary interactions through
activation of reporter gene expression. With the advent of ultrasensitive
mass spectrometric protein identification methods, it is feasible to
identify directly protein complexes on a proteome-wide scale. Here we
report, using the budding yeast Saccharomyces cerevisiae as a test
case, an example of this approach, which we term high-throughput mass
spectrometric protein complex identification (HMS-PCI). Beginning with 10%
of predicted yeast proteins as baits, we detected 3,617 associated proteins
covering 25% of the yeast proteome. Numerous protein complexes were
identified, including many new interactions in various signalling pathways
and in the DNA damage response. Comparison of the HMS-PCI data set with
interactions reported in the literature revealed an average threefold
higher success rate in detection of known complexes compared with
large-scale two-hybrid studies. Given the high degree of connectivity
observed in this study, even partial HMS-PCI coverage of complex proteomes,
including that of humans, should allow comprehensive identification of
cellular networks.
Pseudosubstrate
regulation of the SCF(beta-TrCP) ubiquitin ligase by hnRNP-U.
Davis M, Hatzubai A, Andersen JS, Ben-Shushan E, Fisher GZ, Yaron A,
Bauskin A, Mercurio F, Mann M, Ben-Neriah Y.
Genes Dev 2002 Feb 15;16(4):439-51
The Lautenberg Center for Immunology, The Hebrew University-Hadassah
Medical School, Jerusalem 91120, Israel.
beta-TrCP/E3RS (E3RS) is the F-box protein that functions as the receptor
subunit of the SCF(beta-TrCP) ubiquitin ligase (E3). Surprisingly, although
its two recognized substrates, IkappaB(alpha) and beta-catenin, are present
in the cytoplasm, we have found that E3RS is located predominantly in the
nucleus. Here we report the isolation of the major E3RS-associated protein,
hnRNP-U, an abundant nuclear phosphoprotein. This protein occupies E3RS in
a specific and stoichiometric manner, stabilizes the E3 component, and is
likely responsible for its nuclear localization. hnRNP-U binding was
abolished by competition with a pIkappaB(alpha) peptide, or by a specific
point mutation in the E3RS WD region, indicating an E3-substrate-type
interaction. However, unlike pI(kappa)Balpha, which is targeted by
SCF(beta-TrCP) for degradation, the E3-bound hnRNP-U is stable and is,
therefore, a pseudosubstrate. Consequently, hnRNP-U engages a highly
neddylated active SCF(beta-TrCP), which dissociates in the presence of a
high-affinity substrate, resulting in ubiquitination of the latter. Our
study points to a novel regulatory mechanism, which secures the
localization, stability, substrate binding threshold, and efficacy of a
specific protein-ubiquitin ligase.
The
Vtc proteins in vacuole fusion: coupling NSF activity to V(0) trans-complex
formation.
EMBO J 2002 Feb 1;21(3):259-69
Muller O, Bayer MJ, Peters C, Andersen JS, Mann M, Mayer A.
Friedrich-Miescher-Laboratorium der Max-Planck-Gesellschaft, Spemannstrasse
37-39, D-72076 Tubingen, Germany.
The fusion of cellular membranes comprises several steps; membrane
attachment requires priming of SNAREs and tethering factors by Sec18p/NSF
(N-ethylmaleimide sensitive factor) and LMA1. This leads to trans-SNARE
pairing, i.e. formation of SNARE complexes between apposed membranes. The
yeast vacuole system has revealed two subsequent molecular events:
trans-complex formation of V-ATPase proteolipid sectors (V(0)) and release
of LMA1 from the membrane. We have now identified a hetero-oligomeric
membrane integral complex of vacuolar transporter chaperone (Vtc) proteins
integrating these events. The Vtc complex associates with the R-SNARE Nyv1p
and with V(0). Subunits Vtc1p and Vtc4p control the initial steps of
fusion. They are required for Sec18p/NSF activity in SNARE priming, membrane
binding of LMA1 and V(0) trans-complex formation. In contrast, subunit
Vtc3p is required for the latest step, LMA1 release, but dispensible for
all preceding steps, including V(0) trans-complex formation. This suggests
that Vtc3p might act close to or at fusion pore opening. We propose that
Vtc proteins may couple ATP-dependent NSF activity to a subset of V(0)
sectors in order to activate them for V(0) trans-complex formation and/or
control fusion pore opening.
Michael L. Nielsen, Keiryn
L. Bennett, Brett Larsen, Marc Moniatte, and Matthias
Mann. Peptide
End Sequencing by Orthogonal MALDI Tandem Mass Spectrometry Journal of Proteome Research, ASAP Article
Highly sensitive peptide fragmentation
and identification in sequence databases is a cornerstone of proteomics.
Previously, a two-layered strategy consisting of MALDI peptide mass
fingerprinting followed by electrospray tandem mass spectrometry of the
unidentified proteins has been successfully employed. Here, we describe a
high-sensitivity/high-throughput system based on orthogonal MALDI tandem
mass spectrometry (o-MALDI) and the automated recognition of fragments
corresponding to the N- and C-terminal amino acid residues. Robotic
deposition of samples onto hydrophobic anchor substrates is employed, and
peptide spectra are acquired automatically. The pulsing feature of the
QSTAR o-MALDI mass spectrometer enhances the low mass region of the spectra
by approximately 1 order of magnitude. Software has been developed to
automatically recognize characteristic features in the low mass region
(such as the y1 ion of tryptic peptides), maintaining high mass
accuracy even with very low count events. Typically, the sum of the
N-terminal two ions (b2 ion), the third N-terminal ion (b3
ion), and the two C-terminal fragments of the peptide (y1 and y2)
can be determined. Given mass accuracy in the low ppm range, peptide end
sequencing on one or two tryptic peptides is sufficient to uniquely
identify a protein from gel samples in the low silver-stained range.
Andersen JS, Lyon CE,
Fox AH, Leung AK, Lam YW, Steen H, Mann M, Lamond AI. Directed
proteomic analysis of the human nucleolus. Curr Biol 2002 Jan
8;12(1):1-11 (see also companion article Fox AH et. al Curr. Biol
2002
The nucleolus is a subnuclear organelle
containing the ribosomal RNA gene clusters and ribosome biogenesis factors.
Recent studies suggest it may also have roles in RNA transport, RNA
modification, and cell cycle regulation. Despite over 150 years of research
into nucleoli, many aspects of their structure and function remain
uncharacterized. We report a proteomic analysis of human nucleoli. Using a
combination of mass spectrometry (MS) and sequence database searches,
including online analysis of the draft human genome sequence, 271 proteins
were identified. Over 30% of the nucleolar proteins were encoded by novel
or uncharacterized genes, while the known proteins included several
unexpected factors with no previously known nucleolar functions. MS
analysis of nucleoli isolated from HeLa cells in which transcription had
been inhibited showed that a subset of proteins was enriched. These data
highlight the dynamic nature of the nucleolar proteome and show that
proteins can either associate with nucleoli transiently or accumulate only
under specific metabolic conditions.Conclusions: This extensive proteomic
analysis shows that nucleoli have a surprisingly large protein complexity.
The many novel factors and separate classes of proteins identified support
the view that the nucleolus may perform additional functions beyond its
known role in ribosome subunit biogenesis. The data also show that the
protein composition of nucleoli is not static and can alter significantly
in response to the metabolic state of the cell.
Fox AH, Lam YW, Leung
AK, Lyon CE, Andersen J, Mann M, Lamond AI. Paraspeckles.
A novel nuclear domain. Curr Biol 2002 Jan 8;12(1):13-25
The cell
nucleus contains distinct classes of subnuclear bodies, including nucleoli,
splicing speckles, Cajal bodies, gems, and PML bodies. Many nuclear
proteins are known to interact dynamically with one or other of these
bodies, and disruption of the specific organization of nuclear proteins can
result in defects in cell functions and may cause molecular
disease.Results: A proteomic study of purified human nucleoli has
identified novel proteins, including Paraspeckle Protein 1 (PSP1) (see
accompanying article, this issue of Current Biology). Here we show that
PSP1 accumulates in a new nucleoplasmic compartment, termed paraspeckles,
that also contains at least two other protein components: PSP2 and p54/nrb.
A similar pattern of typically 10 to 20 paraspeckles was detected in all
human cell types analyzed, including primary and transformed cells.
Paraspeckles correspond to discrete bodies in the interchromatin
nucleoplasmic space that are often located adjacent to splicing speckles. A
stable cell line expressing YFP-PSP1 has been established and used to
demonstrate that PSP1 interacts dynamically with nucleoli and paraspeckles
in living cells. The three paraspeckle proteins relocalize quantitatively
to unique cap structures at the nucleolar periphery when transcription is
inhibited.Conclusions: We have identified a novel nuclear compartment,
termed paraspeckles, found in both primary and transformed human cells.
Paraspeckles contain at least three RNA binding proteins that all interact
dynamically with the nucleolus in a transcription-dependent fashion.
Steen H, Kuster B, Fernandez M, Pandey
A, Mann M. Tyrosine
phosphorylation mapping of the epidermal growth factor receptor signaling
pathway. J Biol Chem 2002 Jan 11;277(2):1031-9
Phosphorylation is one of the most
common forms of protein modification. The most frequent targets for protein
phosphorylation in eukaryotes are serine and threonine residues, although
tyrosine residues also undergo phosphorylation. Many of the currently
applied methods for the detection and localization of protein
phosphorylation sites are mass spectrometry-based and are biased against
the analysis of tyrosine-phosphorylated residues because of the stability
and low reactivity of phosphotyrosines. To overcome this lack of sensitive
methods for the detection of phosphotyrosine-containing peptides, we have
recently developed a method that is not affected by the more predominant
threonine or serine phosphorylation within cells. It is based on the
specific detection of immonium ion of phosphotyrosine at 216.043 Da and
does not require prior knowledge of the protein sequence. In this report,
we describe the first application of this new method in a proteomic
strategy. Using anti-phosphotyrosine antibodies for immunoprecipitation and
one-dimensional gel electrophoresis, we have identified 10 proteins in the
epidermal growth factor receptor signaling pathway, of which 8 have been
shown previously to be involved in epidermal growth factor signaling. Most
importantly, in addition to several known tyrosine phosphorylation sites,
we have identified five novel sites on SHIP-2, Hrs, Cbl, STAM, and STAM2,
most of which were not predicted to be phosphorylated. Because of its
sensitivity and selectivity, this approach will be useful in proteomic
approaches to study tyrosine phosphorylation in a number of signal
transduction pathways
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