PTMs - Protein
Phosphorylation
australian proteome
analysis facility
APAF - AUSTRALIA’S PREMIER PROTEOMICS PROVIDER SINCE 1995
APAF’s mission is to assist the scientific community address their protein analysis needs. Australian Government support has enabled APAF
to create a world-leading proteomics analysis facility containing the latest infrastructure for advanced protein analysis. Our strengths in
comparative analysis, along with absolute protein quantitation, have increased our capabilities to deliver leading edge proteomic services.
Phosphoproteomics explained
Protein phosphorylation is a ubiquitous and very
important regulatory post-translational modifications
(PTM). It is estimated to be present on one third of all
proteins. Many critical events involved in cellular response
are mediated by phosphorylation and dephosphorylation,
such as regulation of cell signaling, enzymatic activity,
protein conformational changes, protein-protein
interactions and cellular localisation. Abnormal
phosphorylation is often associated with diseases.
Phosphoproteomics refers to the global analysis
of the phosphate modified proteome. Commonly
phosphorylated amino acids include serine, threonine,
and tyrosine and at APAF we have established methods
to enrich and analyse peptides containing these
modified residues. For phospho-serine/threonine
peptides we use a titanium dioxide based enrichment
prior to high resolution LC/MS-MS. For phosphotyrosine
containing peptides immunoaffinity capture is used.
APAF has found that TiO2 enrichments provide a
solution to detect thousands of phosphopeptides from
complex cell lysates.
We deliver leading
edge proteomic
services
APAF’s evaluation:
• TiO2 enrichment techniques are very effective
to enrich thousands of phosphopeptides from
complex cellular samples
• TiO2 is highly efficient at binding phosphopeptides
and can successfully be employed in combination
with high resolution mass spectrometry
to identify phosphopeptides and analyse
non-phospho fraction for quantitation of
protein levels
• Data can be subsequently processed
with bioinformatics to assign locations of
phosphorylated residues with high confidence
We provide services to:
• Research Institutes
• Universities
• Pharmaceutical R & D
Workflow of phosphoproteome enrichment from cell lysate
KEY
Phosphate
Tryptic peptide
TiO2
TiO2
Non-phospho
(LC-MS/MS)
TiO2
Phosphopeptides
(LC-MS/MS)
CPA cells (derived from Barrett’s oesophagus) treated with
a Polo Like Kinase inhibitor
NR Peptides
(including
Peptides
different
Phospho
identified phosphosites
peptides
% enrichment
Sample
Control A
16112
3830
3136
81.88
Control B
17961
4558
3763
82.56
Drug A
13730
3201
2581
80.63
Drug B
18121
4391
3537
80.55
Amino
Acid site
Ser
Thr
Tyr
Cont A
2984
291
8
Cont B
3586
377
18
Drug A
2432
255
3
Drug B
3324
366
10
Number of
Phospho
Single
Di
Tri or more
Cont A
2345
734
57
Cont B
3323
1095
140
Drug A
1994
554
33
Drug B
2674
786
77
ENQUIRE NOW!
E:[email protected]
T: +61 2 9805 3175
F: +61 2 9805 3101
M: +61 416 250 739
skype. apafbdm
www.apaf.com.au
APAF - Australian Proteome Analysis Facility Ltd. Level 1, 3 Innovation Rd Macquarie University Sydney NSW 2109 Australia
MQ 01-003 APAF PROTEIN PHOSPHORYLATION DRAFT 7.indd 1
7/03/13 12:09 PM
Proteomics
assisting your biological research
APAF works with scientists from small academic laboratories to the largest
pharmaceutical companies in the world. We develop new techniques and work
with state of the art published techniques. Whenever we develop new techniques or
deploy new infrastructure we showcase this in our e-newsletters. Our e-newsletters
and journal publications can be viewed on our website.
Services offered at APAF
Discovery and Comparative Techniques
•
•
•
•
iTRAQTM
Label free LC-MS/MS
Phosphoproteomics
Amino thiols
•
•
•
•
SILAC
Di-sulphide bond analysis
Free amino acids in physiological fluids
DIGE
•
•
•
•
MRM–HR on TripleTOF 5600
Identification of proteins from gel bands
Chemical proteomics
Amino acid analysis for the food industry
Targeted Techniques
•
•
•
•
•
SRM
Multiplex bead based assays
co-IP and Mass spectrometry
N-Terminal Sequencing
Glycan characterisation of antibodies
and recombinant proteins
Other Proteomic Services
• Assay development with HPLC
• Abundant protein depletion
• Proteogenomics –
correcting genome annotations
• Protein purification
• Amino acid analysis
Our State of the Art Infrastructure
•
•
•
•
Thermo Orbitrap Velos Elite with ETD • 3 x AB SCIEX Triple TOFTM 5600 Systems
2 x AB SCIEX 5500 QTRAP
• Thermo Q Exactive
Agilent QTOF 4960
• 14 Mass spectrometers
10 Ultra high pressure
chromatography systems
australian proteome
analysis facility
www.apaf.com.au
APAF - Australian Proteome Analysis Facility Ltd. Level 1, 3 Innovation Rd Macquarie University Sydney NSW 2109 Australia
MQ 01-003 APAF PROTEIN PHOSPHORYLATION DRAFT 7.indd 2
7/03/13 12:09 PM