In vitro sampling and storage of proteins with an ultrafiltration collection device (UCD) and analysis with absorbance spectrometry and SELDI-TOF-MS

Frequent in vivo sampling of blood proteins is often stressful, making it difficult to obtain more than a few samples. As a result, only limited time-profiles can be made. We have developed an ultrafiltration collection device (UCD) for continuous sampling. The UCD consists of a hollow fiber, a coil and a flow creator. Hollow fiber membranes are often hydrophobic and this can result in adsorption of protein and/or peptides, leading to clogged membranes. Adsorption was tested with a hydrophobic and hydrophilic peptide and two biocompatible hollow fibers made from different materials. The hollow fiber made from poly(ethylene) coated with ethylenevinyl alcohol gave near 100% recovery for both peptides. This was in contrast to the poly(sulfone) hollow fiber when sampling the hydrophobic peptide. Filling the coil with various peptide concentrations gave good recovery and insignificant diffusion even after storage for 6 d at 37 degrees C. Continuous pulse-free sampling was tested by vacuum. An average flow rate of 423 +/- 50 nl min(-1) over a period of 4 d was created using S-Monovette. The flow rate gradually declined during this period by <5% every consecutive day. In addition, we also examined a complex sample-serum in the poly(ethylene) hollow fiber. Serum and ultrafiltrate were spotted onto a protein chip and analyzed by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS). Six proteins out of 64 were found to be significantly different between serum and the ultrafiltrate (p < 0.05). The UCD has the potential to be used for in vivo real-time monitoring.     

Harnessing the power of electrophoresis and chromatography: Offline coupling of reverse phase liquid chromatography-capillary zone electrophoresis-tandem mass spectrometry for analysis of host cell proteins in monoclonal antibody producing CHO cell line

Host cell proteins (HCPs) are widely regarded as a critical quality attribute for a biotherapeutic product. Bottom up MS is the present gold standard for HCP analysis but suffers from incomplete protein identification due to complex nature of the HCP mixture and limited separation efficiency of the preceding LC-based systems. In this paper, we present for the first time an application involving use of LC-CE-MS/MS platform for analysis of HCPs. It has been demonstrated that the proposed platform has been able to successfully identify 397 HCPs from the supernatants of recombinant Chinese hamster ovary cells, twice and thrice the number of proteins identified by the state-of-the-art LC-MS/MS (189 HCPs) and CE-MS/MS (128 HCPs) analyses, respectively. Of these, 225 HCPs were unique to the LC-CE-MS/MS approach and were not identified by either LC-MS/MS or CE-MS/MS. It is observed that the LC-CE-MS/MS platform combines the benefits of LC-MS/MS and CE-MS/MS techniques and identifies peptides in a wider range of size, pI, and hydrophobicity. Additionally, LC-CE-MS/MS also identified more HCPs associated with cellular components, molecular functions, biological processes, peptidases, and secretory proteins. The proposed approach would thus be a useful addition in HCP analysis and secretome studies of mAb-producing Chinese hamster ovary cells.     

Identification of 2D-gel proteins: a comparison of MALDI/TOF peptide mass mapping to mu LC-ESI tandem mass spectrometry

A comparative analysis of protein identification for a total of 162 protein spots separated by two-dimensional gel electrophoresis from two fully sequenced archaea, Methanococcus jannaschii and Pyrococcus furiosus, using MALDI-TOF peptide mass mapping (PMM) and mu LC-MS/MS is presented. 100% of the gel spots analyzed were successfully matched to the predicted proteins in the two corresponding open reading frame databases by mu LC-MS/MS while 97% of them were identified by MALDI-TOF PMM. The high success rate from the PMM resulted from sample desalting/concentrating with ZipTip(C18) and optimization of several PMM search parameters including a 25 ppm average mass tolerance and the application of two different protein molecular weight search windows. By using this strategy, low-molecular weight (<23 kDa) proteins could be identified unambiguously with less than 5 peptide matches. Nine percent of spots were identified as containing multiple proteins. By using mu LC-MS/MS, 50% of the spots analyzed were identified as containing multiple proteins. mu LC-MS/MS demonstrated better protein sequence coverage than MALDI-TOF PMM over the entire mass range of proteins identified. MALDI-TOF and PMM produced unique peptide molecular weight matches that were not identified by mu LC-MS/MS. By incorporating amino acid sequence modifications into database searches, combined sequence coverage obtained from these two complimentary ionization methods exceeded 50% for approximately 70% of the 162 spots analyzed. This improved sequence coverage in combination with enzymatic digestions of different specificity is proposed as a method for analysis of post-translational modification from 2D-gel separated proteins.     

The application of 2-D dual nanoscale liquid chromatography and triple quadrupole-linear ion trap system for the identification of proteins

2-D nanoscale LC combined with a triple quadrupole-linear ion trap mass spectrometer was applied to the analysis of a complex peptide mixture. A 2-D dual nanoscale LC-MS/MS system was compared to a conventional one. Peptides were separated with a strong cation exchange (SCX) microcolumn in the first dimension and two C18 nanocolumns were used as second dimension. MS experiments were performed using information-dependent data acquisition, where two precursor ions were selected from an enhanced MS (EMS) or an enhanced multicharged ion (EMC) as survey scan. The major benefit of EMC instead of EMS was a two-fold reduction of the data file and a 15% increase of characterized proteins. The advantage of the 2-D dual nanoscale LC-MS/MS system versus the conventional 2-D nanoscale LC-MS/MS system was reflected in the significant increase of peptides which were successfully identified within the same time frame. The first factor contributing to this increase was that the mass spectrometer was collecting twice the number of relevant MS/MS data. The second factor is the use of twice the number of SCX salt fractions in the first dimension, allowing a better sample fractionation, thereby reducing the number of peptides transferred to the second chromatographic dimension per salt fraction.     

Automated hollow fiber-protected dynamic liquid-phase microextraction of pesticides for gas chromatography-mass spectrometric analysis

Dynamic liquid-phase microextraction (LPME) controlled by a programmable syringe pump was evaluated for extracting pesticides in water prior to GC-MS analysis. A conventional microsyringe with a 1.3-cm length of hollow fiber attached to its needle was connected to a syringe pump to perform the extraction. The microsyringe was used as both the microextraction device as well as the sample introduction device for GC-MS analysis. The attached hollow fiber served as the "holder" and protector" of 3 microl of organic solvent. The solvent was repeatedly withdrawn into and discharged from the hollow fiber by the syringe pump. Pesticides were extracted from 4-ml water samples into the organic solvent impregnated in the hollow fiber. The effects of organic solvents, plunger movement pattern, agitation and extraction time were investigated. Good repeatabilities of extraction performance were obtained, with the RSD values ranging from 3.0% (alachlor) to 9.8% (4-chlorophenol) for the 14 pesticides; most RSD values were under 5.0%. The method provided a 490-fold preconcentration of the target pesticides. The limits of detection were in the range of 0.01-5.1 microg/l (S/N = 3) in the GC-MS selected ion monitoring mode. In addition, sample clean-up was achieved during LPME because of the selectivity of the hollow fiber, which prevented undesirable large molecules from being extracted. A slurry sample (mixture of 40 mg soil/ml of water) containing seven pesticides was extracted using this method which also gave good linearity and precision (most RSDs <7.0%, n = 3).     

Development and application of a new on-line SPE system combined with LC-MS/MS detection for high throughput direct analysis of pharmaceutical compounds in plasma

A technique using a fully automated on-line solid phase extraction (SPE) system (Symbiosis, Spark Holland) combined with liquid chromatography (LC)-mass spectrometry (MS/MS) has been investigated for fast bioanalytical method development, method validation and sample analysis using both conventional C18 and monolithic columns. Online SPE LC-MS/MS methods were developed in the automated mode for the quantification of model compounds (propranolol and diclofenac) directly in rat plasma. Accuracy and precision using online SPE LC-MS/MS with conventional C18 and monolithic columns were in the range of 88-111% and 0.5-14%, respectively. Total analysis cycle time of 4 min per sample was demonstrated using the C18 column. Monolithic column allowed for 2 min total cycle time without compromising the quality and validation criteria of the method. Direct plasma sample injection without on-line SPE resulted in poor accuracy and precision in the range of 41-108% and 3-81%. Furthermore, the increase in back pressure resulted in column damage after the injection of only 60 samples.     

GC—MS analysis of carboxylic acids in petroleum after esterification with fluoroalcohols

The GC–MS characteristics of carboxylic acid esters prepared from fluorine-containing alcohols were compared to those of methyl esters. The GC retention of 2,2,2-trifluoroethyl (TFE) esters was less than, and 2,2,3,3,4,4,4-heptafluoro-1-butyl (HFB) esters was approximately equivalent to that of methyl esters. Mass spectra of TFE and HFB aliphatic esters show significantly more intense molecular and key fragment ions than those of methyl esters. Also, owing to their significantly higher molecular weights, TFE or HFB ester molecular ions and most fragment ions of interest occur at significantly higher m/z values than most potential interfering ions. Data for about 70 individual TFE and HFB esters are reported. Application of the methodology to a petroleum-derived carboxylic acid concentrate resulted in identification of straight chain, isoprenoid, methyl-substituted straight chain (2-, 3-, 5-,10-, 12-positions along chain), and dimethyl-substituted straight chain acids containing from 11 to 22 carbons. Benzoic acid and homologs with up to 3-carbons in alkyl substitutents were minor components in the sample. The procedure provided for forming TFE and HFB esters from free acids requires less time and effort than a previously reported method, while retaining its capability for achieving essentially quantitative conversion. Free hydroxyl groups in alcohols and phenols are converted to trifluoroacetate esters concurrently with formation of TFE/HFB carboxylic acid esters. The reaction products, including compounds with two functional groups (diacids, salicylic acid, etc.), chromatograph well on conventional nonpolar GC stationary phases.     

Analysis of low-abundance proteins using the proteomic reactor with pH fractionation

We developed a new method consisting of the proteomic reactor coupled with step pH fractionation for the analysis of low-abundance proteins from minute amount of sample. These new reactors were implemented using both SAX and SCX materials. The pH fractions from the SAX reactor provided higher peptide and protein identification than SCX reactor and conventional solution digestion. Interestingly, the physical characteristics (pI, molecular weight, missed cleavage site and grand average hydrophobicity (GRAVY) index, and number of acid and basic amino acid) of the peptides obtained from the SAX and SCX proteomic reactors are drastically different. Furthermore, nearly half of the peptides observed from the pH fractionations from the SAX reactor are of low abundance while only 22% low-abundance proteins are observed with conventional in-solution digestion following 2D LC-MS/MS analysis.     

Variations of secretome profiles according to conditioned medium preparation: The example of human mesenchymal stem cell‐derived adipocytes

One challenging point in analyzing cellular secretome collected as conditioned medium is cross‐contamination by cell culture media components, especially bovine serum proteins. A common approach for serum removal is to wash the cells, an alternative is to grow cells using serum‐free conditions. Given that the sample processing may influence the phenotype of cells and thus the secretome, it is important to establish the optimal protocol for each cell type. In this study, we compared two methods for preparing conditioned medium from human adipocytes derived from mesenchymal stem cells. Cells were either washed twice with PBS or cultured the last four days of differentiation in serum‐free adipogenic medium. Gene expression of the cells was evaluated by using real‐time PCR and 1D LC‐MS/MS was used to compare secreted proteins present in the culture supernatants. Surprisingly, results showed significant differences in gene expression patterns of the cells and in protein content of the conditioned media and suggested that PBS washes induced severe modifications of the phenotype of cells and thus changes in protein secretion profiles. These data emphasize the significant variations in protein species related to cell manipulations and underline the importance of procedure optimization prior to any proteomic investigation.     

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.     

In vivo protein sampling using capillary ultrafiltration semi-permeable hollow fiber and protein identification via mass spectrometry-based proteomics

Here, we advanced a novel technique using capillary ultrafiltration (CUF) probes to collect in vivo secreted proteins in the subcutaneous tissue of mouse ear. We fabricated two kinds of CUF probe, one with and one without a semi-permeable membrane hollow fiber. Proteins collected by CUF probes were profiled and identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (MADLI-TOF-MS) and quadrupole time-of-flight tandem mass spectrometry (Q-TOF-MS/MS) without using two-dimensional gel electrophoresis (2-DE) separation. Five proteins including cofilin-1, futuin-A, complement C3, gelsolin, and apolipoprotein C-1 were identified from the sample collected by the CUF probe with a semi-permeable membrane hollow fiber. The presence of well documented secretory proteins supports the efficiency of CUF probes in sampling in vivo secreted proteins. We also found that hemoglobin collected by the CUF probe without a semi-permeable membrane hollow fiber completely masked protein identification by mass spectrometry. The presence of relatively large amounts of hemoglobin in this condition illustrates the necessity of the semi-permeable membrane hollow fiber to the technique of CUF probe in conjunction with mass spectrometry. Also, the technique represents a powerful method for the identification of in vivo secreted proteins and has potential application for in the detection of biomarkers for human diseases.     

Large-scale muLC-MS/MS for silver- and Coomassie blue-stained polyacrylamide gels

2-DE combined with LC-MS/MS has become a routine, reliable protein separation and identification technology for proteome analysis. The demand for large-scale protein identifications after 2-DE separation requires a sensitive and high-throughput LC-MS/MS method. In this report, a simple, splitless, fully automated capillary LC-MS/MS system was described for the large-scale identification of proteins from gels stained with either silver or CBB. The gel samples were digested and peptides were extracted using an in-gel digestion workstation. The peptides were automatically introduced into a capillary column by an autosampler connected to an HPLC pump. A nanoLC pump was then used to deliver the gradient and elute the peptides from the capillary column directly into an LCQ IT mass spectrometer. Neither a peptide trapping setting nor a flow split is needed in this simple setup. The collected MS/MS spectra were then automatically searched by SEQUEST, and filtered and organized by DTASelect. Hundreds of silver-stained or CBB-stained Shewanella oneidensis, Geobacter sulfurreducens, and Geobacter metallireducens proteins separated by denaturing or nondenaturing 2-DE were digested and routinely analyzed using this fully automated muLC-MS/MS system. High peptide hits and sequence coverage were achieved for most CBB-stained gel spots. About 75% of the spots were found to contain multiple proteins. Although silver staining is not commonly thought to be optimal for MS analysis, protein identifications were successfully obtained from silver-stained 2-DE spots detected using methods with and without formaldehyde for protein fixation.     

Continuous sample deposition from reversed-phase liquid chromatography to tracks on a matrix-assisted laser desorption/ionization precoated target for the analysis of protein digests

Peptide mass fingerprinting by matrix-assisted laser desorption/ionization (MALDI)-mass spectrometry (MS) is one of the standard high-throughput methods for protein identification today. Traditionally this method has been based on spotting peptide mixtures onto MALDI targets. While this method works well for more abundant proteins, low-abundance proteins mixed with high-abundance proteins tend to go undetected due to ion suppression effects, instrumental dynamic range limitations and chemical noise interference. We present an alternative approach where liquid chromatography (LC) effluent is continuously collected as linear tracks on a MALDI target. In this manner the chromatographic separation is spatially preserved on the target, which enables generation of off-line LC-MS and LC-MS/MS data by MALDI. LC-MALDI sample collection provides improved sensitivity and dynamic range, spatial resolution of peptides along the sample track, and permits peptide mass mapping of low-abundance proteins in mixtures containing high-abundance proteins. In this work, standard and ribosomal protein digests are resolved and captured using LC-MALDI sample collection and analyzed by MALDI-TOF-MS.     

Rapid, accurate and precise quantitative drug analysis: comparing liquid chromatography tandem mass spectrometry and chip-based nanoelectrospray ionisation mass spectrometry

We have developed a liquid chromatography tandem mass spectrometry (LC-MS/MS) system capable of achieving better than 2% accuracy, routinely over a wide concentration range of 1800 ng mL-1. We demonstrate that the necessary high precision, high accuracy and rapid analysis can be achieved using LC-MS/MS technology. Automated nanoelectrospray ionisation tandem mass spectrometry (nanoESI-MS/MS) technology can be employed to eliminate the chromatographic step completely. In this paper, nanoESI-MS/MS is evaluated and compared directly with LC-MS/MS for the quantitative analysis of two-test analytes, amitriptyline (ATT) and 5-methoxytryptamine (5-MTT), in aqueous/organic mixture. Calibration curves were found to be linear over a wide concentration range of 1800 ng mL-1 for both analytes using LC-MS/MS. Using nanoESI-MS/MS ATT gave a linear response while 5-MTT gave a non-linear response using nanoESI-MS/MS over the same concentration range as in LC-MS/MS. Accuracy and precision values of quality control samples (QCs) at four concentration levels were analysed in replicates of six at each level using 5-MTT and ATT as test analytes for both techniques. The LC-MS/MS system was capable of achieving accuracy levels of 99.50101.96% for ATT and 100.17100.40% for 5-MTT. Accuracy levels using nanoESI-MS/MS were not comparable to LC-MS/MS, they ranged from 90.09100.18% for ATT and 95.95113.55% for 5-MTT. The precision values obtained for nanoESI-MS/MS were in good agreement with those obtained by LC-MS/MS.     

Proteome analysis of tunicamycin-induced ER stress

Endoplasmic reticulum (ER) stress occurs upon increased levels of unfolded proteins and results in activation of cellular responses such as the unfolded protein response (UPR) and ER-associated protein degradation (ERAD). To examine ER stress, we performed a quantitative proteome analysis of human neuroblastoma cells using stable isotope labeling with amino acids in cell culture (SILAC) in combination with SDS-PAGE and LC-MS/MS. Proteins associated with the ER were overrepresented in the dataset of altered proteins. In particular, ER chaperones responsible for protein folding were significantly upregulated in response to ER stress. The important ER stress regulator 78 kDa glucose-regulated protein (GRP-78 or BiP) was highly upregulated together with several proteins that have been found to form a multiprotein complex with BiP including cyclophilin B, DnaJ homolog subfamily B member 11, endoplasmin, hypoxia upregulated protein 1, protein disulfide isomerase and protein disulfide isomerase A4 upon tunicamycin-induced ER stress. Furthermore, seven aminoacyl-tRNA synthetases and five proteins belonging to the Sec61 complex were increased in response to tunicamycin-induced ER stress.     

Targeted tandem mass spectrometry for high-throughput comparative proteomics employing NanoLC-FTICR MS with external ion dissociation

Targeted tandem mass spectrometry (MS/MS) is an attractive proteomic approach that allows selective identification of peptides exhibiting abundance differences, e.g., between culture conditions and/or diseased states. Herein, we report on a targeted LC-MS/MS capability realized with a hybrid quadrupole-7 tesla Fourier transform ion cyclotron resonance (FTICR) mass spectrometer that provides data-dependent ion selection, accumulation, and dissociation external to the ICR trap, and a control software that directs intelligent MS/MS target selection based on LC elution time and m/z ratio. We show that the continuous on-the-fly alignment of the LC elution time during the targeted LC-MS/MS experiment, combined with the high mass resolution of FTICR MS, is crucial for accurate selection of targets, whereas high mass measurement accuracy MS/MS data facilitate unambiguous peptide identifications. Identification of a subset of differentially abundant proteins from Shewanella oneidensis grown under suboxic versus aerobic conditions demonstrates the feasibility of such approach.     

Development of a simple in-vial liquid-phase microextraction device for drug analysis compatible with capillary gas chromatography, capillary electrophoresis and high-performance liquid chromatography

A simple, inexpensive and disposable device for liquid-phase microextraction (LPME) is presented for use in combination with capillary gas chromatography (GC), capillary electrophoresis (CE) and high-performance liquid chromatography (HPLC). 1-4 ml samples of human urine or plasma were filled into conventional 4-ml vials, whereafter 15-25 microl of the extraction medium (acceptor solution) was filled into a short piece of a porous hollow fiber and placed into the sample vial. The drugs of interest were extracted from the sample solutions and into the small volumes of acceptor solution based on high partition coefficients and were preconcentrated by a factor of 30-125. For LPME in combination with GC, the porous hollow fiber was filled with 15 microl n-octanol as the acceptor solution. Following 30 min of extraction, the organic acceptor solution was injected directly into the GC system. For LPME in combination with CE and HPLC, n-octanol was immobilized within the pores of the hollow fiber, while the internal volume of the fiber was filled with either 25 microl of 0.1 M HCl (for extraction of basic compounds) or 25 microl 0.02 M NaOH (for acidic compounds). Following 45 min extraction, the aqueous acceptor solution was injected directly into the CE or HPLC system. Owing to the low cost, the extraction devices were disposed after a single extraction which eliminated the possibility of carry over effects. In addition, because no expensive instrumentation was required for LPME, 10-30 samples were extracted in parallel to provide a high number of samples per unit time capacity.     

Bottom-up proteomics of envelope proteins extracted from spinach chloroplast via high organic content CE-MS

A high organic content CE-MS/MS (HOCE-MS/MS) method was developed for the proteomic analysis of envelope proteins extracted from spinach leaves. Separation was performed in a 1-m long hydroxypropyl cellulose coated capillary, using 8% (v/v) formic acid in 70% (v/v) methanol and 22% water as the BGE. A flow-through microvial interface was used to couple the CE system with an Orbitrap Fusion Lumos mass spectrometer, and field-amplified sample stacking was used to improve the concentration sensitivity. Using this optimized method, 3579 peptides and 1141 proteins were identified using the Proteome Discoverer software with a 1% false discovery rate at the protein level. Relative to conventional aqueous CE, HOCE-MS did a better job of discovering hydrophobic peptides and provided more peptide and protein identifications. Relative to nano-LC-MS, it achieved comparable peptide and protein identification performance and detected peptides not identified by LC-MS: of the full set of peptides identified using the two techniques, 19% were identified only using HOCE-MS. It also outperformed nano-LC-MS with respect to the detection of low molecular weight peptides.     

Determination of catecholamines in pheochromocytoma cell (PC-12) culture medium by microdialysis-microbore liquid chromatography

An in vitro microdialysis system was constructed for the measurement of catecholamines in pheochromocytoma cell culture medium. The novel microdialysis device is composed of a petri dish, a dialysis membrane and two transmission tubes. The dialysis membrane is located in the space of a petri dish such that it is immersed in the culture medium. Catecholamines contained in the culture medium diffused into a designed dialysis membrane with sufficient recovery (about 60%). Dialysates were collected by a sampling loop and introduced by an on-line injector to a microbore liquid chromatographic system for analysis of catecholamines. This assay yielded a detection limit of 0.2–0.5 pg/injection with acceptable intra- and inter-assay reproducibilities in 5 μl of dialysates. To evaluate the on-line microdialysis system, PC-12 cells were cultured in a petri dish within an incubator. The baseline concentration of dopamine in PC-12 cell culture medium was about 0.29 ng/ml which was elevated to 2.43 ng/ml after treatment with 0.5 mM potassium cyanide. In conclusion, the present microassay provides for the sensitive, direct measurement of catecholamines in culture medium while minimizing pretreatment procedures for sample preparation.     

等电聚焦预分离用于肝癌细胞分泌蛋白质组的分级与鉴定

分泌蛋白质组(secretome)是指在特定的时空条件下,细胞、组织等分泌的全部蛋白质。分泌蛋白质组可能包含了大量的疾病诊断生物标志物,因此其相关研究越来越受到重视。分泌蛋白质组的组成高度复杂且浓度范围宽,这对分析方法提出了挑战。建立有效的蛋白质或肽段预分离策略,将有利于分泌蛋白质的高覆盖率鉴定。本研究以肝癌细胞系MHCC97L的无血清培养分泌蛋白质为研究对象,采用一种新型等电聚焦预分离(OFFGEL)系统,考察了肽段水平的分级对蛋白质鉴定结果的影响。结果表明,分离后各馏分中肽段的等电点分布与理论预测基本一致,每个馏分中单独鉴定的肽段比例接近80%,显示了该系统对肽段的高分辨分离能力。结合生物质谱技术,在肝癌细胞分泌系统中鉴定了2995个蛋白质,显示了该系统在复杂体系蛋白质组研究中的应用潜力。