Table 1: Comparison of different proteomics-based techniques

METHOD DESCRIPTION / ADVANTAGES / DISADVANTAGES / SENSITIVITY
2D GEL ELECTROPHORESIS/MASS SPECTROMETRY
  • Separation of complex proteins via 2D gel electrophoresis based charge and size
  • Major protein identification by MS
  • Detects about 2000-2500 spots/gel
/
  • Ability to identify unknown proteins
  • Detects protein modification (phosphorylation and methylation)
  • Used for various biological samples, including tissue, blood and other biological fluids
/
  • Proteins expressed at low abundance may be missed
  • Unsuited for diagnostic application
  • Limited reproducibility and high rate of false identification
  • Limited dynamic
    range
  • semi-quantitative
/
  • Detection sensitivity is in the nanogram range (50 ng/spot for Coomassie Blue; 1 ng/spot for silver stain)
  • Using fluorescent 2D-differential gel electrophoresis (2D-DIGE), sensitivity improves by 10 fold (CyDye label)

LIQUID CHROMATOGRAPHY/MASS SPECTROMETRY
  • LC to separate proteins in a sample, with sequential LC for improved separation efficiency
  • MS to systematically identify the major proteins
  • Detects over 1000 proteins/run
/
  • Ability to identify unknown proteins
  • improved separation efficiency compared to 2D gel
  • Used for various biological samples, including tissue, blood and other biological fluids
/
  • Proteins expressed at low abundance may be missed
  • Unsuited for diagnostic application
  • Limited reproducibility and high rate of false identification
  • Limited dynamic
    range
  • semi-quantitative
/
  • Detection sensitivity is in the nanogram range or ~20 cells
  • 1% false positive rate

PROTEIN ARRAY
  • Individual protein immobilization on a solid-support (glass or membrane)
  • Individual proteins identified by labeled antibodies
  • Detects over 1000 proteins/array
/
  • High sensitivity and specificity
  • Good quantitation range
  • High throughput/density amenable for automation
  • Economical and low sample consumption
  • Lots of data from single experiment
  • Software and hardware tools may be shared with DNA microarray
/
  • Limited protein availability from complex protein production process (expression and purification)
  • Limited access to a large number of affinity antibodies for detection.
/
  • Detection sensitivity is in the ng/ml range

REVERSE PHASE PROTEIN ARRAY
  • Multiple whole-cell or tissue lysate immobilization on individual spots on a solid support (similar to tissue microarray format)
  • Presence of specific proteins are detected by antibody
  • Detects < 100 proteins/array
/
  • Highly sensitive detection of proteins
  • High throughput, i.e. a large number of samples on one slide
  • Minimal sample required
  • Reduced number of antibodies needed to detect protein
/
  • Detection sensitivity may be compromised from loss native protein conformation when surface spotted
  • Limited sensitivity to detect low abundance proteins
  • Specificity may be compromised from non-specific antibody binding (i.e. potential for high background)
  • Limited number of available signaling protein-specific antibodies
/
  • Detection sensitivity is in the picogram range
  • Increased sensitivity
  • Using laser capture microdissection, lysates can be analyzed with as few as 10 cells

ANTIBODY ARRAY
  • Capture antibodies are spotted and fixed on a solid surface
  • Proteins (antigens) are captured on the array surface and detected by a second antibody specific for a different epitopes than capture antibody (sandwich format)
  • Detects < 100 proteins/array
/
  • Highly specific from dual antibody detection
  • Highly sensitive
  • High throughput and amenable for automation
  • Possible to detect protein modifications (phosphorylation, methylation, etc) by modification-specific antibodies
  • Suitable for clinical applications
/
  • Protein complexity and denaturation may affect antigen-antibody interaction
  • Need for high-affinity and specific antibodies
    for capture and detection
  • Limited dynamic
    ranges of 2 or 3 orders of magnitude
/
  • Detection sensitivity is in the low pg/ml range

PATHWAY ARRAY
  • Complex proteins in a sample (cells or tissue) are separated via gel electrophoresis
  • Proteins then transfers to nitrocellular membrane
  • Proteins detected by multichannel immunoblot (similar to Western Blot)
  • Detects up to 300 proteins/run
/
  • Highly sensitive with detection of low abundance proteins
  • Highly specific (as determined by immunoreactivity and size)
  • High accuracy and reproducibility
  • Minimal antibody required for each sample
  • Detects protein modifications (phosphorylation, methylation, etc)
/
  • Limited availability of signaling-related antibodies
  • Relative low through- put (one sample per gel
  • Limited dynamic
    ranges of 2 or 3 orders of magnitude
/
  • Detection limit of 1 ng for each protein with chemiluminescence; 0.1 ng with fluorescence
  • Linear detection range is 100 fold for ECL and 1,000 for fluorescence.

BEAD-BASED ARRAY
  • Either capture antibody or proteins are coated on beads
  • Detection of proteins by labeled antibodies (similar to antibody array or ELISA)
  • Detects 50-100 proteins/run
/
  • Highly sensitive and specific
  • High throughput and amenable for automation
  • Detecst protein modifications (phosphorylation, methylation, etc) by modification specific antibodies
  • Suitable for clinical applications
/
  • Protein complexity and denaturation affecting antigen-antibody interaction
  • Need for high-affinity and specific antibodies for capture and detection
  • Limited dynamic ranges of 2 or 3 orders of magnitude
/
  • Detection limit is sufficient to capture low abundance protein analytes down to the pg/mL range

Supplemental Table 1: Microarray technologies used in genomic and epigenetic analysis:

MICROARRAY / CHARACTERISTIC FEATURE
GENE EXPRESSION ARRAY[1] /
  • Simultaneous monitoring of expression levels for >45,000 transcripts to study the effects of certain treatments, diseases, and developmental stages on gene expression using high-density arrays
  • Does not detect splicing variants as probes are designed to interrogate the 3' end of the transcripts
  • Requires as little as 2 ug of starting mRNA for reverse transcription and labeling

ALTERNATIVE SPLICING ARRAY[2] /
  • Used to assess the expression of alternative splice forms of thousands of genes
  • Exon arrays have a different design that employs probes designed to detect each individual exon for known/predicted genes
  • Used to detect different splicing isoforms
  • Requires as little as 100 ng of starting mRNA

MICRORNA ARRAY[3] /
  • A high-throughput technique to assess cancer-specific expression levels for hundreds of miRNAs in a large sample numbers
  • 500 human miRNAs have been recorded in mirbase (
  • miRNAs are involved in gene expression regulation.

SNP ARRAY[4] /
  • Used to identify single nucleotide polymorphisms among alleles within or between populations
  • Evaluates germline mutations in individuals or somatic mutations in cancers, assessing loss of heterozygosity, or genetic linkage analysis
  • Can measure more than 900,000 SNPs in the whole genome

COMPARATIVE GENOMIC HYBRIDIZATION ARRAY[5] /
  • Used to detect loss, gain and amplification of copy number at the chromosomal level
  • Can detect small gains and losses, e.g. Inter-marker distance of ~100-700 base pairs
  • Combined with SNP array, can be used for genome wide association studies

CHIP-ON-CHIP ARRAY[6] /
  • Combines chromatin immunoprecipitation (chip) with microarray technology ("chip").
  • High throughput (genome-wide) identification and analysis of DNA fragments bound by specific proteins such as histones, transcriptional factors.
  • Used to investigate interaction between protein and DNA
  • Identifies binding sites of DNA-binding proteins in a genome-wide basis.

DNA METHYLATION ARRAY[7] /
  • DNA methylation is an abnormal heritable epigenetic modification process occurring in cancer cells whereby cpg dinucleotides are methylated at the C5 position of cytosine
  • The methylation of the 5’ regulatory regions of genes results in gene silencing
  • Methylated DNA are captured with 5-methlycytidine antibody or methyl binding domain proteins and hybridized to a DNA array which contains 385k to 2.1M probes

Supplemental Table 2: List of antibodies included in the immunoblot array (partial list)

______

Cell signaling:ERK1/2, p-ERK1/2 (Thr202/Tyr204), Akt, p-AKT (Ser473), HGF, HGFR, pHGFR (Y1234/Y1235), IGF, IGFR, TGF, TGFR, Notch 4, Notch 1, p38, p-p38 (Thr180/Tyr182), JNK, p-JNK (Thr183/Tyr185), FGFR, p-FGFR (Tyr653/654), VEGFR, p-VEGFR (Tyr951), PKC, p-PKCalpha (Ser657), p-PKCα/β(Thr638/641), PTEN, p-PTEN (Ser380), PI3K, Ras, Raf, EGFR, p-EGFR (Tyr1068), p-EGFR (Tyr1148), p-EGFR (Tyr1173), Her2, p-Her2 (Tyr1221/1222), PDK1, p-PDK1 (Ser241), mTor, p-mTor (Ser2448), HSP90, NF-kB, IKB, c-Kit, c-Kit (Tyr719), PDGFR, GSK3, beta-catenin, p-be,ta-catenin (Ser33/37/Thr41), stat3, p-stat3 (Ser727), stat5, p-stat5 (Tyr694), smad, p-smad (Ser463/465), CREB, p-CREB (Ser133), Frizzled receptor, APC

Cell Growth/Cell Proliferation: Rb, P21, P27, P15, P16, P18, P19, CHK1, CHK2, DP-1, MDM2, BRCA1, BRCA2, GADD45, 14-3-3β

Cell cycle: CDK2, CDK4, CDK6, CDC2p34, CDC25A, CDC25B, CDC25C,Cyclin B, Cyclin D, Cyclin E, Rb

Invasion/metastasis: VEGF, NF-kappaB, IKK, E-cadherin, N-cadherin, HSP90, TGF-beta, osteopontin, KISS1, KAI1, uPA, uPAR, MMP9, ICAM-1, FAK, EphB2, EphB3

Transcription factor: p-c-Jun, ETS1, c-MYC, E2F-1, GATA, Stat1,p-Stat3,p-Stat5,p-Smad1, p-RB, PR, ERa,ERb

Apoptosis/Autophagy: Bax, FAS, BAD, BCL2, BID, BAK, cleaved Caspase 3, cleaved Caspase 8, cleaved Caspase 9, TRAF, p53, XIAP, NFKB, IKB, Bcl-xL, Smac, LC-3I, LC-3II, Cytochrome C, TNF, AKT1, Survivin, RIP

Angionesis: VEGF, VEGFR, E-cadherin, PDGF, PDGFR, TGF-beta, TGF-beta Receptor, TNF alpha, COX-2, FGF, FGFR, EPO,Ang, Endoglin,Neuropilin, MMP9

DNA repair:P53, ATM, Phospho-ATM (Ser1981), ATR, PCNA, BRCA1, Rad52, TDP1, ERCC1, RCA1, BTG2, CCNH, DNMT1, GADD45A, PTTG1, XRCC5

Epithelial-to-mesenchymal transition/Adhesion: E-cadherin, catenin, Ep-CAM, HCAM, ICAM1, VCAM1

______

Supplemental Table 3: Effect of Cdk6 and XIAP silencing on cell viability, cell cycle distribution and necrosis

Cell Viability (%) / Cell Cycle Distribution (%) / Necrosis (%)
(G0/G1 phase) / (S Phase) / (G2+M Phase)
Negative Control / 100 / 66.37 / 15.9 / 17.76 / 2.14
CDK6 siRNA / 8.26 / 83.81 / 4.6 / 10.89 / 14.46
XIAP siRNA / 14.02 / 71.56 / 6.96 / 11.24 / 31.38


Supplemental Table 4: The expression of signaling transduction proteins in HCCs from 4 patients

SIGNALING PROTEINS / PATIENT SAMPLES
A / B / C / D
Akt / 0 / 2651 / 1724 / 1438
BRCA1 / 0 / 1778 / 0 / 0
cdk6 / 652 / 0 / 0 / 0
cPKCα / 13879 / 462 / 269 / 2058
ERK1/2 / 0 / 2308 / 186 / 0
HIF-3α / 0 / 885 / 0 / 0
p27 / 9104 / 702 / 8470 / 9571
XIAP / 0 / 0 / 2036 / 10094

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