Introduction 1 2 3 8 O 4 9 Materials and methods Subjects 1 10 Table 1 Serum samples in the study  Samples for 2-DE Samples for ELISA Healthy NPC Healthy NPC Lymph node status / Negative Positive / Negative Positive Number 42 27 37 30 27 30 Gender (Male/Female) 22/20 21/6 23/14 15/15 21/6 20/10 Age (±SD) 42 ± 7.9 43 ± 10.9 50.5 ± 11.7 42.5 ± 8.9 43 ± 10.9 47.6 ± 10.2 Depletion of high-abundance proteins ® 11 2D analysis 3 12 In-gel enzymatic digestion 4 3 2 4 3 2 MALDI-TOF-MS identification The peptide mixtures were solubilized with 0.5% TFA, with saturated α-cyano-4-hydroxy-trans-cinnamic (CHCA) solution in 0.1% TFA/50% acetonitrile as the matrix and analyzed using M@LDI R (Micromass, Manchester, UK). Mass spectra were externally calibrated with lock mass 2,465.199 Da and internally calibrated with autodigested peaks of trypsin (MH+: 2,211.105 Da). Protein identification and database searching http://www.matrixscience.com/ P Enzyme-linked immunosorbent assay (ELISA) confirmation ELISAs were conducted to confirm the protein identification and differential expression of sICAM-1, SAA and HSP70 in sera from the three groups. Measurements were done by commercially available ELISA kits (StressXpress Hsp70 ELISA Kit: EKS-700B, Stressgen Biotechnologies, Victoria; High sensitivity sICAM-1 ELISA Kit: BMS241, Bender Medsystems Company, Australian; Human SAA ELISA Kit: EL10015, YES Biotech Laboratories Ltd, Canada), in accordance with the manufacturer’s instructions. All sera were stored at −80°C before they were measured. Both standards and samples were run in duplicate. The main protocols were as follows: monoclonal coating antibody was adsorbed onto microwells; target protein present in the sample or standard bound to antibodies was adsorbed to the microwells; second antibody was added and bound to target protein captured by the first antibody; following incubation unbound enzyme was removed during a wash step; a color reaction was formed and absorbance was measured at 450 nm. The standard curve was used to determine the concentration of target protein in an unknown sample. Immunohistochemistry (IHC) assay 13 14 15 16 Statistical analysis 2 17 t P Results Quantitative comparison and identification of protein spots on 2D gels P  1 2 2 2 3 Fig. 1 a b c  Fig. 2 a 1 2 b P P  Table 2 Identification results of differentially expressed proteins among healthy volunteers, non-LNM NPC and LNM NPC patients a Protein description MSDB ID b c d I e 1 Ovochymase precursor Q7RTY7 ↑ 116 52 124,947/8.67 2 Arachidonate 12-lipoxygenase AAA51587 ↑ 193 68 75,694/5.82 3 sICAM-1 Q99930 ↑ 112 54 4270/6.40 4 Cytochrome P450 Q53EX9 ↑ 207 57 55,635/6.83 5 Serum amyloid A1 protein precursor YLHUS ↑ 170 85 13,524/6.28 6 Hemoglobin beta subunits are s-nitrosylated 1BUWB ↑ 207 55 15,565/6.76 7 E1A 10S protein Q9YLA2 ↑ 143 69 28,493/4.09 8 Lysine-specific histone demethylase 1 O60341 ↑ 172 58 93,358/6.11 9 KIAA1622 protein Q9HCF0 ↑ 78 61 102,351/8.42 10 Heat shock 70 kDa protein (HSP70) Q6G1F9 ↑ 157 53 68,161/4.88 11 Transferrin Q9NQB8 ↓ 167 67 77,050/6.81 12 UPF0366 protein C11orf67 AAD40378 ↓ 104 58 12,798/8.58 13 Transthyretin AAA61181 ↓ 87 69 12,836/5.35 a 1 2 b c d e Fig. 3 P   ELISA confirmation 4 5 P  P  4 5 P  t P  Fig. 4 P P  Fig. 5 P P P  IHC assay 6 3 P  Fig. 6 a b c d e f a b d e  Table 3 Characteristics of NPC patients dependent on ICAM-1 and HSP70 expressions Characteristics Number ICAM-1 expression HSP70 expression Negative Positive (%) P Negative Positive (%) P Gender     Male 41 9 32 (78.0)  0.464 15 26 (63.4)  0.704     Female 16 5 11 (68.8)   5 11 (68.8)  Age Mean (45.4)      <45 years 26 7 19 (73.1)  0.704 9 17 (65.4)  0.945     ≥45 years 31 7 24 (77.4)   11 20 (64.5)  Lymph node status     Positive 30 3 27 (90.0)  0.012 5 25 (83.3)  0.002     Negative 27 11 16 (59.3)   15 12 (44.4)  TNM stage     I + II 24 11 13 (54.2)  0.002 12 12 (50.0)  0.044     III + IV 33 3 30 (90.9)   8 25 (75.8)  Survival rate     Survival 20 6 14 (70) 0.280 6 14 (70) 1.000     Mortality 6 0 6 (100)  1 5 (83.3) Note Discussion 18 19 20 21 22 23 24 25 27 3 8 9 28 29 31 32 33 Of the 13 successfully identified protein spots, we focused on 10 up-regulated proteins in NPC for further validation. We also accumulated certain knowledge about three proteins by literature profiling. Finally, we decided to further investigate sICAM-1, HSP70 and SAA, which seemed more associated with our research interest, using both ELISA and IHC to validate their differential expressions. Intriguingly, most of these identified proteins have been reported to be associated with carcinogenesis and tumor metastasis. Our research is herein chiefly concerned with the functional implications of the three proteins to NPC at the serum level. 13 14 34 35 36 37 38 39 40 41 36 38 39 13 35 42 43 16 15 44 45 46 47 24 26 27 24 48 49 50 51 52 53 54 55 56 57 58 59 60 61 57 58 61 63 in vivo 64 65 59 In conclusion, our serum proteomic analysis, using a comprehensive pretreatment strategy, provides a practical and exemplary tool of screening progression-associated serum proteins in NPC research. After comparing 2D image analyses for healthy volunteers, non-LNM NPC and LNM NPC patients, we successfully identified 13 differentially expressed protein spots. We further explored the differential expressions of three of the proteins, namely, sICAM-1, HSP70 and SAA, by ELISA at serum and IHC at tissue, though more work should be completed to pinpoint the direct correlation between serum level of sICAM and HSP70 and their expression level in the tissues. We suggest that the three proteins may be potential serum biomarkers which can serve as effective target points for early diagnosis and therapy of NPC patients, though further clinical research should be done before the potential come true.