Lower molecular weight (LMW) fraction of plasma proteins includes proteins with less than 30 kDa molecular weight. This is of particular interest in case of cancers as tumor microenvironment has been shown to differentially express the exoproteases leading to cancer-specific proteolytic activity and altered post-translational modifications resulting in production of LMW proteins and peptides from larger molecules (Villanueva et al. 2006). The LMW fraction of plasma largely remains uncharacterized until recently due to the renal clearance limit of 30 kDa and its variability and more importantly due to the pre-fractionation procedures used (Maack et al. 1979; Tirumalai et al. 2003; Adkins et al. 2002).

Partial denaturation of plasma leads to enrichment of LMW fraction and results have supported the identification of proteomic patterns observed in case of breast and ovarian cancers (Merrell et al. 2004; Huang et al. 2005). A number of the potential biomarkers for detection of cancer have been reported in the recent past, majority of these are represented by the LMW proteins such as Fibrinopeptide A for hepatocellular carcinoma (Orvisky et al. 2006), serum amyloid A in renal cancer (Tolson et al. 2004), and kallikreins in prostate cancer. Apart from the intact LMW proteins, a number of proteolytic products of higher MW proteins also contribute to the LMW proteome of plasma. This includes derivatives of complement proteins, IgG, enzymes, transport proteins, and tissue protein derivatives (Fig. 9.1).

Technical issues, particularly reproducibility, have hampered the large-scale analysis of LMW proteome. In the past, removal of high-abundance proteins, centrifugal filtration, and IEF have been employed with adequate success but were not found to be reproducible. Not until recently, albumin was explored as a source for the detection of these LMW proteins due to its properties of carrier protein.

The renal clearance limit of globular proteins in humans is 30 kDa and theoretically any protein/peptide below this limit is rapidly cleared by glomerular filtration in kidneys. The concentration of a biomarker is function of the ratio between biomarker production rate from the tissue and rate of clearance of the carrier protein which in this case is albumin. Consequently, albumin binding amplifies the total biomarker concentration levels measured in serum or plasma. Amplification occurs because the carrier protein acts as a reservoir to accumulate the biomarker over time, as the tissue is continuously producing the biomarker. Thus, a biomarker produced by a small volume of tissue such as the ovary (Petricoin et al. 2002a, b), prostate (Petricoin et al. 2002c), or breast (Li et al. 2002), at a low concentration (e.g., one femtomole per day), can accumulate to a concentration of one picomole in plasma because it binds with albumin that has long half-life of 6 days. Figure 9.2 shows the mechanism of amplification of LMW proteins by binding to albumin which functions as reservoir for the accumulation and amplification of low mass biomarkers produced during cancer progression.

Kinetics of albumin association to the lower molecular weight biomarkers have revealed that binding to albumin may affect the protein’s availability, detection, quantization, and overall utility as a biomarker (Mehta et al. 2003). Additionally, the presence of unbound and bound states of proteins in plasma can affect both the clearance of free-state proteins/peptides and also their detection by current proteomic approaches. Further, pattern of albumin bound proteins is distinct from those bound to other proteins (Mehta et al. 2003). Novel protein hits continue even over five iterations indicative of vast repertoire of albumin-associated proteins. It is therefore imperative that examination of the LMW species bound to albumin may contain important diagnostic information for neoplastic transformation.

Albumin and its bound proteins and peptides are referred to as albuminome that is a naturally occurring sub-proteome in blood. A robust, optimized, and reproducible chemical method for the isolation of albumin from serum based on classical Cohn fractionation has recently been used (Fu et al. 2005). Chemical fractionation eliminated the problem of protein carryover in subsequent experiments and can be extended to any volume of plasma and was compatible with both 2DE and LC-MS/MS. From the reported presence of 120 nonredundant proteins in the albumin-enriched fraction – albuminome – majority (75 %) were extracellular proteins, whereas the minority (25 %) were cellular proteins (Fu et al. 2005). This latter category contains transport proteins, enzymes, and protease inhibitors. Although many of the proteins present in the albumin-enriched fraction are high-abundance proteins, such as α1-antitrypsin and transferrin, lower-abundance proteins such as fibrinogen and angiotensinogen as well as cellular proteins including actin and tubulin were identified (Fu et al. 2005).

Comparison with existing literature revealed that 26 of the 35 identified albumin-associated proteins have been previously reported potential biomarkers (Anderson 2005; Polanski and Anderson 2006). Of these, nine were observed only in the albumin-enriched fraction and not in the albumin-depleted fraction (Sheng et al. 2006). Proteins in this category are α-2HS-glycoprotein, apolipoprotein A-I, ceruloplasmin, inter-α-trypsin inhibitor H4, kininogen, apolipoprotein C-III, fibrinogen, retinol-binding protein, and β-thromboglobulin. Furthermore, five of the nine albumin-bound potential biomarkers were observed only in the albumin-enriched fraction (α-2HS-glycoprotein, apolipoprotein A-I, apolipoprotein C-III, retinol-binding protein, and ceruloplasmin) and were observed in intact form. Since intact potential biomarkers were observed exclusively in the albumin-enriched fraction, therefore it is crucial to know in which fraction a potential biomarker will be located after sample fractionation with the caveat being whether all proteins present in the original sample will be represented in the depleted fraction. This makes the albumin-enriched fraction, and particularly the albuminome, an important sub-proteome in biomarker discovery.