Analysis of human serum by liquid chromatography-mass spectrometry: improved sample preparation and data analysis

Discovery of biomarkers is a fast developing field in proteomics research. Liquid chromatography coupled on line to mass spectrometry (LC-MS) has become a powerful method for the sensitive detection, quantification and identification of proteins and peptides in biological fluids like serum. However, the presence of highly abundant proteins often masks those of lower abundance and thus generally prevents their detection and identification in proteomics studies. To perform future comparative analyses of samples from a serum bank of cervical cancer patients in a longitudinal and cross-sectional manner, methodology based on the depletion of high-abundance proteins followed by tryptic digestion and LC-MS has been developed. Two sample preparation methods were tested in terms of their efficiency to deplete high-abundance serum proteins and how they affect the repeatability of the LC-MS data sets. The first method comprised depletion of human serum albumin (HSA) on a dye ligand chromatographic and immunoglobulin G (IgG) on an immobilized Protein A support followed by tryptic digestion, fractionation by cation-exchange chromatography, trapping on a C18 column and reversed-phase LC-MS. The second method included depletion of the six most abundant serum proteins based on multiple immunoaffinity chromatography followed by tryptic digestion, trapping on a C18 column and reversed-phase LC-MS. Repeatability of the overall procedures was evaluated in terms of retention time and peak area for a selected number of endogenous peptides showing that the second method, besides being less time consuming, gave more repeatable results (retention time: <0.1% RSD; peak area: <30% RSD). Application of an LC-MS component detection algorithm followed by principal component analysis (PCA) enabled discrimination of serum samples that were spiked with horse heart cytochrome C from non-spiked serum and the detection of a concentration trend, which correlated to the amount of spiked horse heart cytochrome C to a level of 5 pmol cytochrome C in 2 microl original serum.     

Enrichment of low-abundant serum proteins by albumin/immunoglobulin G immunoaffinity depletion under partly denaturing conditions

We present a simple protocol for affinity depletion to remove the two most abundant serum proteins, albumin and immunoglobulin G (IgG). Under native conditions, albumin/IgG were efficiently removed and several proteins were enriched as shown by two-dimensional electrophoresis (2-DE). Besides that, partly denaturing conditions were established by adding 5 or 20% acetonitrile (ACN) in order to disrupt the binding of low-molecular-weight (LMW) proteins to the carrier proteins albumin/IgG. 2-DE results showed that the total number of detected LMW proteins increased under denaturing conditions when compared to native conditions. Interestingly, the presence of 5% ACN in serum revealed better enrichment of LMW proteins when compared to 20% ACN condition. Seven randomly distributed spots in albumin/IgG depleted serum samples under 5% ACN condition were picked from the 2-DE gels and identified by mass spectrometry (MS). The intensity of five LMW protein spots increased under denaturing conditions when compared to native conditions. Three of the seven identified spots (serum amyloid P, vitamin D-binding protein, and transthyretin) belong to a group of relatively low-abundant proteins, which make up only 1% of all serum proteins. The method presented here improves the resolution of the serum proteome by increasing the number of visualized spots on 2-D gels and allowing the detection and MS identification of LMW proteins and proteins of lower abundance.     

Enrichment of low molecular weight serum proteins using acetonitrile precipitation for mass spectrometry based proteomic analysis

A rapid acetonitrile (ACN)-based extraction method has been developed that reproducibly depletes high abundance and high molecular weight proteins from serum prior to mass spectrometric analysis. A nanoflow liquid chromatography/tandem mass spectrometry (nano-LC/MS/MS) multiple reaction monitoring (MRM) method for 57 high to medium abundance serum proteins was used to characterise the ACN-depleted fraction after tryptic digestion. Of the 57 targeted proteins 29 were detected and albumin, the most abundant protein in serum and plasma, was identified as the 20th most abundant protein in the extract. The combination of ACN depletion and one-dimensional nano-LC/MS/MS enabled the detection of the low abundance serum protein, insulin-like growth factor-I (IGF-I), which has a serum concentration in the region of 100 ng/mL. One-dimensional sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the depleted serum showed no bands corresponding to proteins of molecular mass over 75 kDa after extraction, demonstrating the efficiency of the method for the depletion of high molecular weight proteins. Total protein analysis of the ACN extracts showed that approximately 99.6% of all protein is removed from the serum. The ACN-depletion strategy offers a viable alternative to the immunochemistry-based protein-depletion techniques commonly used for removing high abundance proteins from serum prior to MS-based proteomic analyses.     

A comparison of depletion versus equalization for reducing high-abundance proteins in human serum

In this work three methods to diminish the content of most highly abundant proteins in human serum have been studied and compared. Protein depletion with ACN or DTT and protein equalization with the ProteoMiner^? (PM) have been assessed by 1‐D gel electrophoresis and MS. After treatment 5, 18 and 9 major proteins within the 20 most abundant proteins in serum were identified for the ACN, DTT and PM methods, respectively. The ACN method was efficient for depleting high molecular weight proteins, over 75 KDa, resulting in 10±4% (n=3) of the total protein content remaining in the depleted serum. In addition, 75% of the proteins belonging to the group of the 20 most abundant proteins were not detected, making this depletion strategy a cheap alternative to expensive commercial tools regularly used for removing high abundance proteins from serum. The ACN extract was found rich in apolipoproteins. The dithithreitol method promotes the precipitation of proteins rich in disulfide bonds, mainly albumin, with 73±7% (n=3) of the total protein content remaining in the depleted serum, which was found rich in immunoglobulins. The PM method compresses the dynamic range of the serum proteins, rendering an extract containing 16±2% (n=3) of the total initial protein content. The extract was found to be rich in both apolipoproteins and immunoglobulins. As a general rule the DTT and PM methods provide a compression of the dynamic range of serum protein concentrations while the ACN method allows an effective depletion of the protein fraction above 72 KDa.     

Identification of candidate biomarkers in ovarian cancer serum by depletion of highly abundant proteins and differential in‐gel electrophoresis

Ovarian cancer is the fifth leading cause of cancer death for women in the US, yet survival rates are over 90% when it is diagnosed at an early stage, highlighting the need for biomarkers for early detection. To enhance the discovery of tumor‐specific proteins that could represent novel serum biomarkers for ovarian cancer, we depleted serum of highly abundant proteins which can mask the detection of proteins present in serum at low concentrations. Three commercial immunoaffinity columns were used in parallel to deplete the highly abundant proteins in serum from 60 patients with serous ovarian carcinoma and 60 non‐cancer controls. Medium and low abundance serum proteins from each serum pool were then evaluated by the quantitative proteomic technique of differential in‐gel electrophoresis. The number of protein spots that were elevated in ovarian cancer sera by at least twofold ranged from 36 to 248, depending upon the depletion and separation methods. From the 33 spots picked for MS analysis, nine different proteins were identified, including the novel candidate ovarian cancer biomarkers leucine‐rich α2 glycoprotein‐1 and ficolin 3. Western blotting validated the relative increases in serum protein levels for three of the proteins identified, demonstrating the utility of this approach for the identification of novel serum biomarkers for ovarian cancer.     

A rapid sample pretreatment protocol: improved sensitivity in the detection of a low-abundant serum biomarker for prostate cancer.

We have developed a rapid immunoglobulin G (IgG) and a human serum albumin (HSA) depletion protocol. We depleted both HSA and IgG (> 97%) separately, and in a single procedure. The method is specific and reproducible (RSD < 1.0%), and substantially lowered the detection limit of prostate-specific antigen, a prostate cancer biomarker. The method can be applied to other biomarkers and proteomic studies. Interestingly, the depletion of HSA might not be blankly as beneficial as widely portrayed. Our study suggests the depletion of IgG to be more beneficial than albumin depletion.     

A modified protein precipitation procedure for efficient removal of albumin from serum

Proteomic analysis of sera and the quest for identifying serum proteins as disease markers have often been hampered by the predominance of several highly abundant proteins including albumin and immunoglobulins. Prior albumin depletion so as to enrich for otherwise undetectable serum components is therefore a prerequisite in mining the serum proteome. In the course of evaluating several available methods and commercial kits, we have been able to refine the albumin depletion protocols and establish a modified albumin removal method using trichloroacetic acid (TCA)/acetone. Changes in major protein bands were monitored by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (1-D SDS-PAGE) and used as the first screening strategy to evaluate and optimize for the precipitation experimental conditions. Our method showed better performance in efficiency, specificity, and costs in comparison with two commercially available albumin removal kits, and provides a simple pre-fractionation step for the proteomic analysis of serum biomarkers. Albumin isolated by the modified method is in the native state. Our method may offer a rapid method for purifying serum albumin in large scale.     

Sample complexity reduction aids efficient detection of low‐abundant proteins from human amniotic fluid

Working with biological fluids poses a challenge of visualizing proteins present in lower concentrations. This study describes a batch‐mode chromatographic method for the fractionation of human amniotic fluid (AF). This method is easy to use with minimal sample quantity, resin volume and sample processing time. For albumin depletion, two methods were evaluated. The results demonstrated that specific depletion of albumin, using affinity‐ligand‐based resin, is more efficient than the conventional dye‐based method. The albumin‐depleted human AF was fractionated by strong anion‐exchange resin in spin devices, for sample, complexity reduction and enrichment of low‐abundant proteins. Analysis of four eluate fractions generated after this step shows enrichment of few low‐abundant proteins. Two novel low‐abundant proteins, Rab GDP dissociation inhibitor β and peptide methionine sulfoxide reductase, were identified from human AF. α‐1‐B Glycoprotein was successfully identified by this strategy, whereas the published literature reports that it was not identified by strong anion‐exchange FPLC followed by SDS‐PAGE. Therefore, the current method has distinct advantages over the conventional column‐based chromatography. This study also reports altered expression of some proteins in Rh‐isoimmunized AF samples in comparison with normal AF.     

Characterization of the human serum depletome by label-free shotgun proteomics

While it is known that immunoaffinity depletion of abundant proteins in serum removes additional proteins beyond those targeted, there has been little characterization of the co-depleted proteins in the high abundant fraction, which we refer to here as the "depletome". We present evidence of co-depletion of non-targeted proteins in human serum using a top-20 immunodepletion column, as shown by label-free liquid chromatography mass spectrometry (LC-MS(E)) profiling. This led to identification of 147 proteins which were specific for this fraction and comprised proteins with functions predominantly in binding and transport of nucleotides, metal ions, carbohydrates and lipids. These results suggest that further studies on this commonly ignored serum fraction may provide new insights into clinical proteomics.     

High‐abundance protein depletion: Comparison of methods for human plasma biomarker discovery

Affinity depletion of abundant proteins from human plasma has become a routine sample preparation strategy in proteomics used prior to protein identification and quantitation. To date, there have been limited published studies comparing the performance of commercially available depletion products. We conducted a thorough evaluation of six depletion columns using 2‐DE combined with sophisticated image analysis software, examining the following criteria: (i) efficiency of high‐abundance protein depletion, (ii) non‐specific removal of other than the targeted proteins and (iii) total number of protein spots detected on the gels following depletion. From all the products investigated, the Seppro IgY system provided the best results. It displayed the greatest number of protein spots on the depleted plasma gels, minimal non‐specific binding and high efficiency of abundant protein removal. Nevertheless, the increase in the number of detected spots compared with the second best performing and cheaper multiple affinity removal column (MARC) was not shown to be statistically significant. The ProteoPrep spin column, considered to be the “deepest” depletion technique available at the time of conducting the study, surprisingly displayed significantly fewer spots on the flow‐through fraction gels compared with both the Seppro and the MARC. The spin column format and low plasma capacity were also found to be impractical for 2‐DE. To conclude, we succeeded in providing an overview of the depletion columns performances with regard to the three examined areas. Our study will serve as a reference to other scientists when deciding on the optimal product for their particular projects.     

The application of ultra-performance liquid chromatography/tandem mass spectrometry to the detection and quantitation of apolipoproteins in human serum

The detection and quantitation of apolipoproteins, important markers for coronary heart disease, in serum by liquid chromatography/tandem mass spectrometry (LC/MS/MS) using multiple reaction monitoring (MRM) is reported. A tryptic digest of depleted human serum was analysed by nanoflow LC/MS/MS at a flow rate of 300 nL/min and several apolipoproteins (Apo), including Apo A1, A2, A4, C1, C2, C3, D, F and M, were successfully identified. The analysis of the same depleted serum digest by ultra-performance (UP)LC/MS/MS operating at 700 microL/min resulted in comparable sensitivity and selectivity to the nanoflow method, but with a dramatic ( approximately 20-fold) reduction in run time. The potential of UPLC/MS/MS for the rapid quantitation of proteins in biological matrices by representative tryptic peptides was further investigated using Apo A1 and its corresponding stable isotopically labelled tryptic AQUA peptide (DYVSQFEGSALGK). A set of serum-based Apo A1 calibrators from a clinical analyser kit were digested without depletion following the addition of the AQUA peptide and analysed using UPLC/MS/MS. A linear calibration curve was generated from peak area ratios to the labelled peptide with a coefficient of correlation of 0.9989. Standard curves were also generated for other apolipoproteins together with Apo B100, Apo E, lecithin cholesterol acyltransferase and albumin, which were also detected in the standards. The concentration of Apo A1 in five fresh undepleted human serum samples and a quality control (QC) sample were determined using both the UPLC/MS/MS method and a clinical analyser. Results were comparable and the quantitative study, involving 80 injections which took hours rather than days to complete, demonstrates the high-throughput potential of UPLC/MS/MS to quantify multiple serum proteins without the need for antibodies, and thus provide an alternative to the use of clinical analysers for serum protein biomarkers.     

Searching for serum tumor markers for colorectal cancer using a 2‐D DIGE approach

Identification of specific protein markers for colorectal cancer (CRC) could provide a basis for its early diagnosis and detection, as well as clues to the molecular mechanisms governing cancer progression. In the present study, 2‐D DIGE coupled with MS was used to screen for biomarker candidates in the serum proteome of ten human CRC samples and ten healthy control samples. After pooling identical amounts of serum proteins (based on total protein concentration), albumin/IgG was depleted under partially denaturing conditions. Subsequently, the serum samples were labeled with three different CyDyes, and separated by 2‐D DIGE. After analysis with the biological variation analysis module of the DeCyder software, only three spots were found to be significantly elevated in all patient groups (with ratios from 1.52 to 9.08), whereas five spots were significantly down‐regulated in patients (with ratios from ?1.23 to ?10.21) (t‐test; p<0.05). Finally, two potential biomarkers, Transaldolase 1 and thyroid receptor interactor, were chosen for validation and analysis by ELISA with the serum of 30 CRC patients and 30 healthy controls. The serum levels of the two proteins correlated well with the 2‐D DIGE results. Thus, 2‐D DIGE approaches show great promise for biomarker discovery in CRC.     

Micro-high-performance liquid chromatography platform for the depletion of high-abundance proteins and subsequent on-line concentration/capturing of medium and low-abundance proteins from serum. Application to profiling of protein expression in healthy and osteoarthritis sera by 2-D gel electrophoresis

In this investigation, an integrated microcolumn-based fluidic platform for the simultaneous depletion of high-abundance proteins and the subsequent on-line concentration/capturing of medium- and low-abundance proteins from human serum has been introduced. The platform consists of on-line coupling of tandem affinity micorcolumns to an RP microcolumn to achieve first the depletion of high-abundance proteins by the tandem affinity microcolumns followed by the concentration and capturing of medium- and low-abundance proteins by the RP microcolumn. The tandem affinity microcolumns are based on macroporous monoliths characterized by their relatively high permeability in pressure-driven flow while the RP microcolumn is packed with polymeric particles with an average particle diameter of 20 microm giving rise to a very little back pressure, thus allowing fast flow velocity across the coupled columns format and consequently short processing time of serum samples prior to analysis by 2-DE. The microcolumn-based fluidic platform was applied to serum samples from osteoarthritis (OA) donors before and after soy protein (SP) supplementation, and from healthy donors, and the resulting depleted serum samples from high-abundance proteins were profiled for protein expression by 2-DE. In general, the protein expression was lower in serum of the same OA patient after soy treatment than before soy treatment. Several proteins were down-regulated after soy treatment with transthyretin being the most affected by the SP supplementation. In addition, with respect to serum from healthy donors, the sera from OA patients showed difference in proteins expression.     

Biomarker discovery in breast cancer serum using 2-D differential gel electrophoresis/ MALDI-TOF/TOF and data validation by routine clinical assays

In the present study, we used 2-D differential gel electrophoresis (2-D DIGE) and MS to screen biomarker candidates in serum samples obtained from 39 patients with breast cancer and 35 controls. First, we pooled the serum samples matched with age and menopausal status. Then, we depleted the two most abundant proteins albumin and IgG by immunoaffinity chromatography under partly denaturing conditions in order to enrich low-abundance proteins and proteins with low molecular weight. Concentrated and desalted samples were labeled with three different CyDyes including one internal standard, pooled from all the samples, and separated with 2-D DIGE in triplicate experiments. Biological variations of the protein expression level were analyzed with DeCyder software and evaluated for reproducibility and statistical significance. The profile of differentially expressed protein spots between patients and controls revealed proapolipoprotein A-I, transferrin, and hemoglobin as up-regulated and three spots, apolipoprotein A-I, apolipoprotein C-III, and haptoglobin alpha2 as down-regulated in patients. Finally, routine clinical immunochemical reactions were used to validate selected candidate biomarkers by quantitative determination of specific proteins in all individual serum samples. The serum level of transferrin correlated well with the 2-D-DIGE results. However, the serum levels of apolipoprotein A-I and haptoglobin could not be detected with the clinical routine diagnostic tests. This demonstrated an advantage 2-D DIGE still has over other techniques. 2-D DIGE can distinguish between isoforms of proteins, where the overall immunochemical quantification does fail due to a lack of isoform-special antibodies.     

Bifunctionalized SBA-15 as a novel micropipette tip sorbent for selective removal and enrichment of biomolecules

Selective enrichment or removal of specific proteins prior to analysis can minimize nonspecific interferences as well as information loss, which has been an important issue in current proteomics fields. In this work, two kinds of mesoporous silica SBA-15 (mean pore diameter of 5 nm and 7 nm), bifunctionalized with quaternary ammonium and C18 groups via silylanization reaction, was used to investigate the adsorption behavior for different proteins (bovine serum albumin (BSA), ovalbumin (OVA), hemoglobin (Hb), lysozyme (Lys) and cytochrome c (cyt c)). As possessing anion exchange (quaternary ammonium) groups, the bifunctionalized SBA-15 showed selective adsorption of the negative charged proteins, BSA and OVA. Based on these properties, sequential fractionation based on different SBA-15 materials as micropipette tip sorbents was developed for sample preparation prior to protein analysis. As expected, after the sequential treatment of the sample, the detection signal of the high abundant BSA was significantly decreased and that of the low abundant cyt c was obviously enlarged, which solved the problem that low abundant protein was suppressed by adjacent high abundant protein. The sample preparation technique significantly improved protein identification and this sequential fractionation approach could be potentially applied to extend information on the protein identification for biological samples with a wide dynamic range.     

Discovery of the serum biomarker proteins in severe preeclampsia by proteomic analysis

Preeclapsia (PE) is a severe disorder that occurs during pregnancy, leading to maternal and fetal morbidity and mortality. PE affects about 3-8% of all pregnancies. In this study, we conducted liquid chromatography- mass spectrometry/mass spectrometry (LC-MS/MS) to analyze serum samples depleted of the six most abundant proteins from normal and PE-affected pregnancies to profile serum proteins. A total of 237 proteins were confidently identified with <1% false discovery rate from the two groups of duplicate analysis. The expression levels of those identified proteins were compared semiquantitatively by spectral counting. To further validate the candidate proteins with a quantitative mass spectrometric method, selective reaction monitoring (SRM) and enzyme linked immune assay (ELISA) of serum samples collected from pregnant women with severe PE (n = 8) or normal pregnant women (n = 5) was conducted. α2- HS-glycoprotein (AHSG), retinol binding protein 4 (RBP4) and α-1-microglobulin/bikunin (AMBP) and Insulin like growth factor binding protein, acid labile subunit (IGFBP-ALS) were confirmed to be differentially expressed in PE using SRM (P<0.05). Among these proteins, AHSG was verified by ELISA and showed a statistically significant increase in PE samples when compared to controls.     

Characterization of insulin adsorption in the presence of albumin by time-of-flight secondary ion mass spectrometry and X-ray photoelectron spectroscopy

With a view to develop an encapsulation membrane for a bioartificial pancreas, we have studied the adsorption of insulin and human serum albumin (HSA) on it. The aim of this study was to determine the possibility of insulin detection on a polycarbonate membrane surface in the presence of HSA, an abundant blood protein. The first step of the work consisted in the identification of time-of-flight secondary ion mass spectrometry (ToF-SIMS) and X-ray photoelectron spectroscopy (XPS) specific signals for insulin and albumin. For this purpose, adsorption isotherms in physiological conditions (pH = 7.2, T = 37 degrees ) were established for the two proteins by looking at the SIMS intensity variations of the characteristic protein and substrate fragments when increasing the protein concentration in the solution. The CHS+ ToF-SIMS fragment and the S2p XPS peak were identified as representative insulin signals. The second step of the work consisted in performing simultaneous adsorption of the two proteins with increasing insulin concentration. We observed an increase of the insulin signal in ToF-SIMS and XPS for insulin concentration beyond 5 microg/mL. Principal component analysis (PCA) of the ToF-SIMS results permits us to obtain information about the protein layer composition. The results show that at low relative insulin concentration in solution, the mixed adsorbed layers are enriched in insulin compared to the solution.     

Low-molecular-weight human serum proteome using ultrafiltration, isoelectric focusing, and mass spectrometry

Identification of the serum proteome is a daunting analytical task due to the complex nature of the sample which has an extremely large dynamic range of protein components. This report addresses this issue by using centrifugal ultrafiltration to enrich the low-molecular-weight (LMW) serum proteome while decreasing the amount of abundant high-molecular-weight proteins. Reduction of the complex nature of the sample was achieved by fractionation of the LMW serum proteins using solution-phase isoelectric focusing (IEF). Multiple enzyme digestions are performed and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Analysis of the tandem mass spectra resulted in the identification of 262 proteins belonging to LMW serum proteome. Our results demonstrate the effectiveness of this methodology to isolate and identify LMW proteins with improved confidence in the MS data acquired. In addition, our methodology can be combined with other multidimensional chromatography techniques performed on the peptide level to increase the number of identified proteins.     

功能化介孔硅材料的合成及其对富含裸露组氨酸蛋白质的选择性吸附

通过自由基聚合反应合成了具有高比表面积的介孔硅(PPOSS),并对其表面进行修饰和铜离子(Cu~(2+))的固定,最终得到功能化介孔硅材料(PPOSS-IDA-Cu~(2+)),然后利用PPOSS-IDA-Cu~(2+)中的Cu~(2+)与蛋白质裸露组氨酸的螯合作用,选择性吸附富含裸露组氨酸的蛋白质。对牛血红蛋白、牛血清白蛋白、肌红蛋白和溶菌酶的吸附结果表明,所合成的PPOSS-IDA-Cu~(2+)材料对富含组氨酸的牛血红蛋白有较好的吸附选择性和较大的吸附容量(3 150mg/g),并且有望与其他材料联合使用,以检测到更多相对丰度较低的蛋白质,丰富人类蛋白质组学信息,为疾病的临床诊断及病理研究提供帮助。     

Combinatorial peptide ligand library plasma treatment: Advantages for accessing low‐abundance proteins

Depletion of major blood proteins is one of the most promising approaches to accessing low‐abundance biomarkers. This study compared the use of combinatorial peptide ligand library (CPLL) and albumin and immunoglobulins (IgGs) depletion technology for accessing these low‐abundance proteins in plasma using 2‐DE in an acidic restricted pH range (4–7). Compared with native plasma, both techniques enlarge the visibility of other proteins than albumin and IgG, but there were marked differences in their composition. An increase of the number of spots was detected compared with native plasma (157 spots) with 427 and 557 spots, respectively, detected with albumin and IgG depletion, and CPLL treatment. We selected 70 spots to be identified by MALDI‐TOF related to their absence in the 2‐D gels from native or albumin and IgG‐depleted plasma. The 42 spots identified corresponded to 24 different proteins, with more than half of the proteins which did not belong to the major plasma proteins. CPLL treatment allowed the accessibility to proteolytic fragments obtained from major plasma proteins. We found a large superiority of the CPLL approach over the albumin and IgG depletion process. These findings show the utility of depleting major blood proteins to be able to access low‐abundance proteins and the potential of CPLL to select and identify candidate biomarkers in clinical studies.