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The identification of circulating biomarkers holds great potential for non invasive approaches in early diagnosis and prognosis, as well as for the monitoring of therapeutic efficiency.1-3 The circulating low molecular weight proteome (LMWP) composed of small proteins shed from tissues and cells or peptide fragments derived from the proteolytic degradation of larger proteins, has been associated with the pathological condition in patients and likely reflects the state of disease.4,5 Despite these potential clinical applications, the use of Mass Spectrometry (MS) to profile the LMWP from biological fluids has proven to be very challenging due to the large dynamic range of protein and peptide concentrations in serum.6 Without sample pre-treatment, some of the more highly abundant proteins obscure the detection of low-abundance species in serum/plasma. Current proteomic-based approaches, such as two-dimensional polyacrylamide gel-electrophoresis (2D-PAGE) and shotgun proteomics methods are labor-intensive, low throughput and offer limited suitability for clinical applications.7-9 Therefore, a more effective strategy is needed to isolate LMWP from blood and allow the high throughput screening of clinical samples.
Here, we present a fast, efficient and reliable multi-fractionation system based on mesoporous silica chips to specifically target and enrich LMWP.10,11 Mesoporous silica (MPS) thin films with tunable features at the nanoscale were fabricated using the triblock copolymer template pathway. Using different polymer templates and polymer concentrations in the precursor solution, various pore size distributions, pore structures, connectivity and surface properties were determined and applied for selective recovery of low mass proteins. The selective parsing of the enriched peptides into different subclasses according to their physicochemical properties will enhance the efficiency of recovery and detection of low abundance species. In combination with mass spectrometry and statistic analysis, we demonstrated the correlation between the nanophase characteristics of the mesoporous silica thin films and the specificity and efficacy of low mass proteome harvesting. The results presented herein reveal the potential of the nanotechnology-based technology to provide a powerful alternative to conventional methods for LMWP harvesting from complex biological fluids. Because of the ability to tune the material properties, the capability for low-cost production, the simplicity and rapidity of sample collection, and the greatly reduced sample requirements for analysis, this novel nanotechnology will substantially impact the field of proteomic biomarker research and clinical proteomic assessment.
As shown in Figure 1, in this study we fabricated a series of mesoporous silica thin films with a variety of nanotextures and comprehensively explored their use in selective capturing and enriching LMW peptides and proteins from human serum. Figure 2a and b show the MS spectra of the unprocessed serum sample for peptides in the range of 900 to 10,000 Da and for proteins in the range of 3,000 ~ 70,000 Da respectively. These spectra illustrate the signal suppression in the LMW region due to the presence of well ionized, highly abundant, high molecular weight (HMW) proteins such as Albumin. Figure 2c and d depict the MS spectra of the serum sample after fractionation by the MPS L121 (pore size, 6 nm). The majority of the large molecules have been depleted, resulting in a significant enrichment of the LMW components. As a control, the same serum sample was applied onto a nonporous pure silica surface to evaluate the specificity of MPS thin films for LMWP recovery. As can be seen in Figure 2e and f, there was no significant harvesting of peptides or proteins from the nonporous silica. Thus it can be concluded that it was the mesoporous architecture and not the silica surface affinity that constitutes the predominate factor in the enrichment of LMWP.
Precisely controlled variations in pore size can be achieved through the use of copolymers with differing hydrophobic block lengths. By using the designed proteins and peptides mixture, the effect of pore size on the LMW peptide and protein recovery efficacy was investigated using MPS thin films prepared from four Pluronic surfactants (F127, P123, L121, and L121 plus swelling agent) with different volume ratios of the hydrophilic and hydrophobic components to form pore sizes of 3.7 nm, 5.2 nm, 7.4 nm, and 9.0 nm respectively. This range of pore sizes led to the recovery of a different repertoire of peptides and proteins from the same serum sample via size and shape exclusion (Figure 3). The protein spectra at high molecular weight demonstrate the molecular cut-off of each type of chip. In addition to the size-dependant depletion of HMW proteins, the on-chip fractionation of the standards solution displays a differential and selective enrichment of LMW species associated with the pore sizes. The two-way hierarchical clustering presented in Figure 3b shows the LMW standards enrichment pattern obtained with the different MSC. Even if all the peptides are below the molecular cut-off of the chips, there is a positive correlation between the pore sizes and the molecular weight of the trapped species. The MSC with large pores, up to 9 nm, preferentially harvest bigger peptides, while smaller peptides are recovered more efficiently by the chips with smaller pores. The structural transformation of the mesoporous arrangement was carried out by tuning the concentration of the template polymer. Increasing the concentration of the template polymer resulted in a reduced interfacial curvature between the phases of the water, the copolymer, and the silicate, consequently initiating the interrelated progression from a spherical to a cylindrical structure. Pluronic F127, with its high molecular weight, possesses this high degree of structural periodicity. By increasing the concentration of F127 in starting solution, different MPS thin film periodic nanostructures can be obtained from 3D nanostructure to 2D nanostructure. The 3D cubic and honeycomb hexagonal nanostructures, possessing more desirable nanopore interconnectivity and more accessible nanopore morphology, exhibit superior performance in selectively enriching LMW peptides than the 2D hexagonal structure, even though they share similar pore size distributions and the same molecular cut-off for serum fractionation (Figure 4). We also streamlined the conjugation of organo-silane on MPS chips by introducing oxygen plasma ashing to pretreat the chip surface. In order to qualitatively study the electrostatic effect on selective on-chip enrichment, we use the proteins and peptides mixture. MS analysis of the proteomic standards solution fractionated on MPS chips prepared with L121 and conjugated with the chemical functional groups is presented in Figure 5. The positively charged and negatively charged the peptides and LMW proteins are captured on the anionic and the cationic chips respectively. The quantitative comparison of multiple MPS chips in recovering the peptides with positive net charge is displayed in Figure 5a. The chips with negative charge and the chips without any modification (with a minor negative charge originally) exhibit significantly higher enrichment for those peptides than the chips modified with APTES (-NH2). Conversely, the positively charged MPS chips possess exceptional capability to recover those peptides with negative net charge, as demonstrated in Figure 5b. While α-Endorphin does not show a significant change due to its PI around 6.
Figure 1. Principle of MPS chips fractionation and LMW enrichment. After sample spotting on the surface, LMW proteins and peptides are trapped into the pores while the larger species remained outside the pores and are removed during the washing steps. The enriched fractions are then eluted and analyzed by MALDI.
Figure 2. Peptide enrichment using the mesoporous silica thin film chips. MALDI MS profiles in both the low mass range (900 to 10 000 Da) and the high mass range (3000 to 70 000 Da) before (a,b) and after (c,d) serum processing on the mesoporous silica thin films (L121, 6 nm). The molecular recovery is significantly reduced when using blank nonporous silica surfaces (e,f).
Figure 3. Molecular cut-off and size-dependent enrichment of the MPS chips. (a) Magnified view of the MALDI spectra demonstrating the characteristic molecular cut-off of each MPS chips correlating to the pore size. (b) Two-way hierarchical clustering of the peptide mix features among the different chips. The intensity of the red or yellow color indicates the relative peptide concentration. Larger pores enhanced the harvesting of larger peptides (from 3600 to 8500 Da), while the small peptides (from 900 to 3500 Da) were preferentially recovered from the chips with smaller pores.
Figure 4. Physical characterizations of MPS thin films and selective recovery with different nanostructures. XRD patterns (a, b, c), TEM (inset a, b, c), Pluronic F127 at different concentrations in the precursor solution: 4.0×10-3 M (a), 6.0×10-3 M (b), and 8.0×10-2 M (c). (d) Bar graph of the intensity of detection illustrating the selective peptides recovery on 3D cubic and 3D hexagonal F127 proteomic chips (Cub and Hex, respectively). The different structural modifications present a selective enrichment.
Figure 5. Charge-specific recovery for the chips with different surface functions. Bar graph of the MS intensity of detection of selectively captured peptides on the functionalized chips. According to their iso-electric point, the peptides are positively or negatively charged at pH 7.0. (a) Positive peptides ((1) des-Arg1-Bradykinin, (2) Bradykinin, (3) substance P-amide, (4) Neurotensin, (5) ACTH(1-17), (6) ACTH(7-38)) are specifically enriched on the negatively charged surfaces. (b) Negative peptides ((7) Glu1-fibrinopeptide B, (8) α-endorphin, (9) ACTH(18-39), (10) insulin, (11) EGF, (12) insulin-like GFII) are specifically enriched on the positively charged surfaces. Please click here to see a larger version of this figure.
Figure 6. On-chip stabilization of fractionated serum. (a) Representative MALDI profiles of LMW peptides and proteins eluted immediately after serum fractionation (top) or after 3 wk of on-chip storage at room temperature (bottom). (b) From top to bottom: Linear regression analysis of average intensities of detected MS peaks in each replicate compared to replicate 1 for freshly fractionated serum and fractionated serum after 3wk MPS chips storage at room temperature. Equation, CV and coefficient of determination (R2) are indicated. Please click here to see a larger version of this figure.
Evidence is mounting that the low molecular-weight region of the circulatory proteome is a rich source of diagnostic biomarkers for the early detection of disease. In this technology, we presented a series of mesoporous silica chips with different pore sizes, pore structures and modifications to selectively enrich peptides and low molecular weight proteins. To evaluate protein stability, the MPS chips were incubated with human serum, dried after washing, and stored for 3wk at room temperature. The protein/peptide patterns obtained were comparable with those of freshly fractionated serum (Figure 6a), as confirmed by the results of the statistical analysis showed in Figure 6b. The variability of the peak signals measured by the average CV was estimated at 12.7% for crude serum and at 14.2% for the fractionated samples. The marginal variations could be due to the internal variability of the MALDI instrument and suggested that the on-chip pretreatment and storage did not induce any significant alteration of the MS protein profiles. The same experiment has been performed on non porous silicon. The dried serum recovered after storage on silicon surface was fractionated on the MPS chips before MALDI analysis. The poor MS profile obtained demonstrated the stabilization advantage of the mesoporous surface. In analogy with previously postulated mechanisms, we hypothesize that the LMW species trapped inside the nanopores were preserved from degradation through the size exclusion of proteases, or by steric inhibition of their proteolytic activity in the confined space of the nanopores. The MPS chip based method could be a powerful tool in the peptide and LMW protein profiling of complex biological fluids study. The MPS chips are inexpensive to manufacture and allow for scaled up production to attain the simultaneous processing of a large number of samples, providing advantageous features for exploratory screening and biomarker discovery.
We have nothing to disclose.
This work was funded by Alliance of NanoHealth Pre-center Award (W81XWH-11-2-0168) and Texas Center for Cancer Nanomedicine (1U54CA151668-01).