Serum is a complex body fluid, containing a large diversity of proteins. More than 10000 different proteins are present in the human serum and many of them are secreted or shed by cells during different physiology or pathology processes [41] . Serum is expected to be an excellent source of protein biomarkers because it circulates through, or comes in contact all tissues. Consequently, serum proteomics has raised great expectations for the discovery of biomarkers to improve diagnosis or classification of a wide range of diseases, including cancers [42] . However, serum has been termed as the most complex human proteome [43] with considerable differences in the concentrations of individual proteins, ranging from several milligrams to less than one pictogram per milliliter [44] . The analytical challenge for biomarker discovery arises from the high variability in the concentration and state of modification of some human plasma proteins between different individuals [45] . Albumin is a protein of very high abundance in serum (35-50 mg/mL) that would be a prime candidate for complete selective removal prior to performing a proteomic analysis of lower abundance proteins. Thus, removal of albumin from serum may also result in the specific removal of low abundance cytokines, peptide hormones, and lipoproteins of interest. Immunoglobulins, and antibodies are also abundant proteins in serum that function by recognizing "foreign" antigens in blood and initiating their destruction [46] . The presence of higher abundance proteins interferes with the identification and quantification of lower abundance proteins (lower than ng/mL in serum). Complexity and dynamic range of protein concentrations can be addressed with a combination of prefractionation techniques that deplete highly abundant proteins and fractionate. Heparin chromatography coupled with protein G appears to be an efficient and economical strategy to pretreat serum for serum proteomics [47] . Protein prefractionation by immunodepletion and reversed-phase separation of the depleted plasma on mRP-C18 column provide methods compatible with LC-MS-based analysis. A polyclonal antibody-based system to rapidly deplete multiple high abundant proteins in serum, plasma, CSF, and other biological fluids. Individual antibody materials are mixed in selected percentages and packed into a column format. Albumin can be removed by immunoaffinity columns [48] , isoelectric trapping [49] , dye-ligand chromatography [50] , and peptide affinity chromatography [51] . Another approach involves the removal of IgG by affinity chromatography using immobilized protein A or protein G [52] . A recently developed depletion method that mixes 6 high-specificity polyclonal antibodies (MARS) to remove the top 6 proteins in a single purification step is commercially available [53] . Human-14 multiple affinity removal column depletes the top 14 abundant proteins from human serum, plasma, CSF, and other biological fluids. To address 2D limitations several types of mass spectrometry, in conjunction with various separation and analysis methods, are increasingly being adopted for proteomic measurements [54] . In contrast, 2D-PAGE analysis, SELDI-TOF MS is a rather new method which is especially valuable for the identification of serum-derived biomarkers [55] . This method is based on ProteinChip Arrays which carry various chromatographic properties, such as anion exchange, cation exchange, and hydrophilic or hydrophobic surfaces [56] . For the analysis of serum, only 5-10 μL of serum sample is applied to these surfaces; after washing off unbound material, the protein fingerprint can be determined and visualized by time-of-flight mass spectrometry. The advantages of this method are the low amount of sample necessary for analysis, its speed, and high throughput capability. Many different groups have used this method and related methods based on prefractionation of serum proteins by beads and subsequent MALDI analysis for the identification of biomarkers in serum, urine, pancreatic juice, and other biological fluids [57] . The necessity of this removal or separation is also illustrated that many proteins found useful as biomarkers [58] . Different fractionation steps (such as electrophoresis, SELDI, and liquid chromatography) have been developed to reduce the complexity of serum proteome and to allow the detection and the identification of single proteins [59] . 2DE and MALDI MS had applied to identify candidate biomarkers at early and late stages of lung cancer disease. This method identified 46 proteins in tumor bearing mice this included disease regulated expression of orosomucoid-8, a-2-macroglobulin, apolipoprotein-A1, apolipoprotein-C3, glutathione peroxidase-3, plasma retinol-binding protein, and transthyretin [60] . Recently 1065 proteins were identified by stable isotope labeled proteome (SILAP) standard coupled with extensive multidimensional separation with tandem mass spectrometry of which 121 proteins were present at 1.5-fold or greater concentrations in the sera of patients with pancreatic cancer [61] . Specimen collection (Blood, serum, plasma samples) is an integral component of clinical research. Access to high-quality specimens, collected and handled in standardized ways that minimize potential bias or confounding factors, is key to the "bench to bedside" aim of translational research [62] . Variables that may impact analytic outcomes include (1) the type of additive in the blood collection tubes; (2) sample processing times or temperatures; (3) hemolysis of the sample; (4) sample storage parameters; (5) the number of freeze-thaw cycles [63, 64] .