Download In vivo protein biotinylation for identification of organ

In vivo protein biotinylation for identification of organspecific antigens accessible from the vasculature
Jascha-N Rybak, Anna Ettorre, Brigitte Kaissling,
Raffaella Giavazzi, Dario Neri & Giuliano Elia
Nature Method Vol 2,No 4, 291
The endothelium is highly dynamic structure, morphologically and functionally adapted to meet the unique
needs of the underlying tissue.
Tumors requires a blood supply for expansive growth, an observation that has stimulated a profusion of
research on tumor angiogenesis.
Blood vessels of tumors have multiple structure and functional abnormalities. Their unusual leakiness,
potential for rapid growth and remodeling, and expression of distinctive surface molecules not only are
responsible for mediating hematogenous spread of tumor cells and maintaining the unusual
microenvironment of tumors but also are key to the efficacy of targeted tumor therapy.
Genes expressed in human tumor endothelium. Science 289, 1197 (2000)
Abnormalities of basement membrane on blood vessels and endothelial sprouts in tumors. American
Journal of Pathology 163,1801 (2003)
Subtractive proteomic mapping of the endothelial surface in lung and solid tumors for tissue-specific
therapy. Nature 429, 629 (2004)
Direct proteomic mapping of the lung microvascular endothelial cell surface in vivo and in cell culture.
Nature Biotechnology 22, 985 (2004)
Injections of F9 murine teratocarcinoma
Or tumor cells were inoculated in the left kidney
Blood components were washed away with
perwarmed PBS supplemented with 10% dextran
40 as plasma expander.
The perfusion solution contained 1mg/ml sulfoNHS-LC-biotin in PBS and 10% dextran 40
Quenching step : 50 mM Tris solution in PBS
Histochemical analysis of organ and tumor sections
skeletal muscle
tongue
Saline
Sulfo-NHSLC-biotin
kidney
Saline
Sulfo-NHSLC-biotin
F9 tumor
Anti-CD31
liver
Chromatographic elution profiles of tryptic peptides from the healthy and
tumor-bearing kidenys of RENCA mice
It is showed that consistent differences among normal and tumor-bearing kidneys
Subcellular localization of protein identified
Cell surface proteins and extracellular matrix proteins were present in all organs
examined, in proportions that ranged cumulatively between 20% and 50%.
Western blot analysis of organ-specific markers
Anti-Ksp cadherin: anti-kidney-specific cadherin 16
Anti-calcium channel L-type DHPR alpha 1 subunit
Anti-calcium channel, voltage-gated alpha2/delta1 subunit
Anti-protein tyrosine kinase 7
Anti-T lymphoma invasion and metastasis inducing protein 1
Immunofluorescence detection of organ-specific makers
kidney
Anti-Ksp cadherin
Skeletal
muscle
Anti-calcium channel L-type
DHPR alpha 1 subunit
Kidney-specific cadherin 16 was detected in lateral cell membranes bordering the intercellular spaces
between the tubular epithelial cells.
Calcium channel L-type DHPR alpha 1 subunit is seen in the transverse tubule system, surrounding
the muscle fibers.
This method offers several potential advantages:
1.
Is compatible with the use of strong anionic detergents (such as SDS), which are indispensable for the
solubilization of most membrane proteins and extracellular matrix components
2.
The chemical properties of the reactive organic molecule used in the perfusion reaction can be changed
to influence which structures can be labeled and recovered in vivo. Indeed, the approach is not limited
to the use of biotin; any other reactive molecule for which a high-affinity reagent is available could be
considered.
3.
The proteomics analysis described in this article is easy to implement, is sensitive and allows the
hundreds of different accessible proteins in the tissues of interest.
Transketolase, a thiamine diphosphate-dependent enzyme linking the nonoxidative branch of pentose
phosphate pathway to the glycolytic pathway, has been shown to control in vivo tumor growth in mice with
Ehrlich ascites tumor and to be overexpressed in highly metastatic adenoid cystic carcinoma cell lines as
compared with their poorly metastatic counterparts.
Recently, acetyl CoA carboxylase 265 has been identified as a partner of the protein encoded by the breast
cancer susceptibility gene BRCA1. This report show that acetyl CoA carboxylase 256 is either more
abundant, more easily accessible, or both in the tumors tested than in other normal tissues, suggesting that
it could be used as antigen for ligand-based tumor targeting applications.
A non-negligible fraction of identified proteins were intracellular proteins or serum components. Sulfo-NHSLC-biotin has been shown in in vivo studies to be capable of passing through cell membrane and to label
intracellular proteins, in addition to surface proteins, but disulfide-linked botin derivatives such as sulfoNHS-SS-botin yield a more specific labeling of membrane proteins. However, use of sulfo-NHS-SS-biotin
did not improve recovery of membrane proteins, possibly owing to in vivo instability.
Serum components were observed at higher frequency in solid tumor.
 blood coagulation products have long been known as components of provisional stroma that sustains
tumor cell growth.
 transient thrombotic events in some tumor blood vessels or insufficient perfusion of the tumor mass
Quantitative phosphoproteome analysis using a dendrimer
conjugation chemistry and tandem mass spectrometry
W Andy Tao, Bernd Wollscheid, Robert O’Brien, Jimmy K Eng, Xiao-jun Li, Bernd Bodenmiller
Julian D Watts, Leroy Hood & Ruedi Aebersold
Nature Method Vol 2,No 8, 591
Reversible protein phosphorylation has a vital role in regulating many complex biological processes
such as cellular growth, division and signaling.
Phosphorylated proteins and, in particular, the dynamically phosphorylated forms of signaling
proteins, are often of low abundance.
In contrast to identification of phosphorylation sites within these phosphoproteins is far more difficult.
To identify the sites of phosphorylation, it is essential to have an efficient strategy for the enrichment of
actual phosphopeptides.
Several approaches have been explored to date for the selective isolation of phosphopeptides; the
most notable of these are either affinity- or chemical derivatization -based.
Phosphotyrosine antibody:
Temporal analysis of phosphotyrosine-dependent signaling networks by quantitative proteomics.
Nature Biotechnology 22, 1139 (2004)
It is typically limited to specific amino acid sequences or phosphotyrosine-containing species.
Immobilized metal ion affinity chromatography:
Phosphoproteome analysis by mass spectrometry and its application to Saccharomyces cerevisiae.
Nature Biotechnology 20, 301 (2002)
This approach has yield considerable results, its selectivity appears to be dependent on several
operational parameters including the metal ion, the ligand and the stationary phase used.
The method appears to be highly dependent on the type of resin and pH condition used for binding,
and elution and favors peptides with multiple phosphorylation sites.
Chemical modification:
A systematic approach to the analysis of protein phosphorylation. Nature Biotechnology 19, 375 (2001)
Typically involve several derivatization steps, and to have had limited applications for the analysis of
phospho-peptides within complex mixture.
1. Amino protection: t-butyl dicarbonate (tBoc)
to eliminate the potential for intra- and
intermolecular condensation
2. Condensation reaction: Carbodiimide catalyzes
Condensation reactions between the peptides
and excess amine to form amide and
phosphoamide bonds.
3. Phosphate regeneration: Free phosphate
groups are regenerated by brief acid
hydrolysis.
4. Condensation and reduction: A
carbodiimide-catalyzed condensation reaction
attaches cystamine to the phosphate group.
Reduction of the internal disulfide of cystamine
next generates a free sulfhydryl group.
Nature Biotechnology 19, 375 (2001
N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide; EDC
5. Solid phase capture: The recovered
phosphopeptide are attached to a solid phase
by reacting the free sulfhydryl groups in the
peptides with iodoacetyl groups immobilized
on the glass beads.
6. Phosphopeptide recovery:
Phosphopeptides are recovered by cleavage
of phosphoamidate bonds using TFA at a
concentration that also removes the tBoc
protection groups.
Schematic illustration of three-step procedure to chemically isolate phosphopeptide
1. Mixtures of peptides are converted to the corresponding methyl esters to protects carboxylate groups.
2. Tagging and isolating phosphopeptides: the methylate peptides are combined and subjected to a one pot
reaction in the presence of carbodiimide (EDC), imidazole and a dendrimer. Phosphate groups are readily
activated using EDC and imidazole and react with excess amines on the dendrimer to form phosphoramidate
bonds.
3. Covalently bound phosphopeptides are readily isolated from nonphosphopeptides using size selective
methods such as a simple membrane-based filter device (5 kDa).
4. Phosphopeptides are detached from the dendrimer through a brief acid hydrolysis of the phosphoramidate
bonds and isolate using the same membrane-based filter device.
Isolation and tagging of phosphopeptides, and validation with beta-casine digests.
100 pmole tryptic digestion of -casine
FQS*EEQQQTEDELQDK (2159.8 m/z)
sample recovery efficiency:
10 pmloe tryptic peptide from casine were labeled with methyl-d0 and -d3 esterification.
Methyl-d0-esterified peptides were subjected to an enrichment step
The recovered methyl-d0 phosphopeptide was add
into the original methyl-d3 peptide mixtures
final yield > 35% of the starting material
The phosphorylated peptide was detected as the most prominent peak in the mass spectrum, with little
contamination from nonphosphopeptides or other contaminants.
Quantification of phosphopeptides in standard protein mixtures
Two mixtures consisting of equalmolar quantities of ovalbumin, BSA, beta-lactoglobulin, lysozyme,
beta-casine and apomyoglobin (10 pmole each)
Each protein mixture was digested and either methyl-d0 or methyl-d3 esterified.
Seven carboxylic groups
FQS*EEQQQTEDELQDK (2159.8 m/z)
Methyl-d0 and -d3 esterification
21Da (10.5 Da for doubly charged )
Light/heavy ratio : 0.89
1091 m/z
1081 m/z
Sensitivity of this method for quantitative analysis
500 pmole of BSA digest was spiked with 1 pmole of signle phosphopeptide
Phosphorylate angiotensin II (DRVY*IHPF)
Mixture was divided equally, and each sample was either methyl-d0 or -d3 esterified.
Enrichment
LTQ (quadrupole linear ion trap mass spectrometer)
10 fmole of phosphopeptides
Analyses of tyrosine phosphorylation sites in human T cells
Stimulation of T cells via cell-surface receptors triggers the activation of the TCR and then activates
intracellular networks of effector molecules.
Dynamic serine/threonine/tyrosine phosphorylation and dephosphorylation of signaling
intermediates by kinase and phosphatase, respectively, is critical for the regulation of the T cell
signaling network.
To fully understand T-cell response, it is necessary to pinpoint temporal phosphorylation and
dephosphorylation events in the course of the response of T cells to various stimuli.
Jurket T cells
Pervanadate (inhibitor of tyrosine phosphatase )
2 min
10 min
Immuno-affinity enrichment (phosphotyrosine antibody : 4G10)
sample was either methyl-d0 or -d3 esterified.
Enrichment
uLC-MS/MS
97 tyrosine phosphoproteins
75 tyrosine phosphorylation sites
80 serine/threonine phosphorylation sites
Our two-step approach of enriching tyrosinephosphorylated proteins by immuno-affinity
selection followed by chemical enrichment of
phosphopeptides led to the MS detection of
all known tyrosine phosphorylated residues in
the (ITAMs) of the TCR’s CD3 chians upon
pervanadate treatment in a single experiment.
Quantitative phosphopeptide analysis
In this case, the ratio (light:heavy) was 2.1, indicating that after a 10-min treatment with pervanadate,
phosphorylation on two tyrosine residues of this peptide was increased twofold compared to the 2-min
treatment.
Abundance ratios of tyrosine phosphopeptides isolated from T cells
treated with pervandate over 10 min or 2 min.
10 min : Light
2 min : Heavy
Ratios: light/heavy
Tyrosine phosphorylation increased with increasing time of pervandate
treatment. This is indicated by frequent observation of abundance
ratios larger than 1.0 for phosphopeptides isolated after treatment for
10 min and 2 min, respectively.
This paper describe an alternative general chemical strategy for the enrichment and subsequent mass
spectrometric analysis of phosphopeptides, which represents a major advance compared to their
previously reported methodology.
This improvement arises primarily from directly capturing phosphopeptides in a single-step reaction with a
primary amine-containng solution polymer (in this case, a Generation-5 polyamidoamine dendrimer) rather
than the complicated chemical transformation described previously.
A dendrimer is a perfectly branched, unimolecular solution polymer that has functional groups only at its
surface. (polylysine abs polyallylamine may also be suitable for this application)
Such soluble reagents permit faster reactions than the chemically functionalized controlled pore glass
beads used previously, owing to their higher capacity and the homogenous nature of the reaction medium.
Using this approach, the authors identified and quantitated the relative changes in over 150 sites of
phosphorylation from Jurket T cells treated with the phosphotase inhibitor.
The true value of this work is in the author’s effective adaptation of techniques from other nonbiologically
oriented field to solve a biochemical problem. Specifically the use of solution polymers, such as
dendrimers, as auxiliaries in organic reactions has a long-established history, but this is the first report of
their effective use in a proteomics application.