Multidimensional Separation Research Articles

An Ion Mobility Assisted Data Independent LC-MS Strategy for the Analysis of Complex Biological Samples

The use of gas-phase ion mobility separation in LC-MS based bottom-up proteomic experiments is described. A data-independent acquisition approach was employed in combination with one and two dimensional reversed phase liquid chromatography separations. The ion mobility component in the acquisition schema affords additional acquisition specificity and improved peptide resolution in terms of system peak capacity. The additional acquisition specificity was mainly achieved by means of the combined retention and drift time association of product to precursor ions and chemical noise reduction. Experimental data from replicate experiments were utilized to determine the rate of increase for the number of detected ions and deconvoluted accurate mass – retention time pairs and protein and peptide identifications as a measure of peak capacity by varying mass spectral resolution, consideration of multidimensional separations and the incorporation of ion mobility into the analytical schema. The presented data reveals a high degree of reproducibility at the ion detection, peptide and protein level, when analyzed under identical conditions, with a concurrent average increase in the number of identified peptides and proteins of 160% in the instance of mobility assisted acquisitions. Two-dimensional separations independently improved the peptide and protein identification rates by another 260% and 320%, respectively. Collectively, both separation techniques, reversed phase chromatography and ion mobility, increased the peak capacity and number of peptides and proteins significantly. The applicability of the technique and instrument platform for qualitative and quantitative proteomics studies will be demonstrated.

The emerging field of chemo‐ and pharmacoproteomics

The emerging field of chemo- and pharmacoproteomics studies the mechanisms of action of bioactive molecules in a systems pharmacology context. In contrast to traditional drug discovery, pharmacoproteomics integrates the mechanism of a drug's action, its side effects including toxicity, and the discovery of new drug targets in a single approach. Thus, it determines early favorable (e.g. multiple kinase target in cancer drugs) and unfavorable (e.g. side effects) polypharmacology. Target profiling is accomplished using either active site-labeling probes or immobilized drugs. This strategy identifies direct targets and has in fact enabled even the determination of binding curves and half maximum inhibitory concentrations of these targets. In addition, the enrichment greatly reduces the complexity of the proteome to be analyzed by quantitative MS. Complementary to these approaches, global proteomics profiling studying drug treatement-induced changes in protein expression levels and/or post-translational modification status have started to become possible mostly due to significant improvements in instrumentation. Particularly, when using multidimensional separations, a considerable proteome depth of up to 10000 proteins can be achieved with current state-of-the-art mass spectrometers and bioinformatics tools. In summary, chemo- and pharmacoproteomics has already contributed significantly to the identification of novel drug targets and their mechanisms of action(s). Aided by further technological advancements, this interdisciplinary approach will likely be used more broadly in the future.

The Application of SILAC Mouse in Human Body Fluid Proteomics Analysis Reveals Protein Patterns Associated with IgA Nephropathy

Body fluid proteome is the most informative proteome from a medical viewpoint. But the lack of accurate quantitation method for complicated body fluid limited its application in disease research and biomarker discovery. To address this problem, we introduced a novel strategy, in which SILAC-labeled mouse serum was used as internal standard for human serum and urine proteome analysis. The SILAC-labeled mouse serum was mixed with human serum and urine, and multidimensional separation coupled with tandem mass spectrometry (IEF-LC-MS/MS) analysis was performed. The shared peptides between two species were quantified by their SILAC pairs, and the human-only peptides were quantified by mouse peptides with coelution. The comparison for the results from two replicate experiments indicated the high repeatability of our strategy. Then the urine from Immunoglobulin A nephropathy patients treated and untreated was compared by this quantitation strategy. Fifty-three peptides were found to be significantly changed between two groups, including both known diagnostic markers for IgAN and novel candidates, such as Complement C3, Albumin, VDBP, ApoA,1 and IGFBP7. In conclusion, we have developed a practical and accurate quantitation strategy for comparison of complicated human body fluid proteome. The results from such strategy could provide potential disease-related biomarkers for evaluation of treatment.

Prefractionation of Intact Proteins by Reversed-Phase and Anion-Exchange Chromatography for the Differential Proteomic Analysis of Saccharomyces cerevisiae

The need for multidimensional separations for bottom-up proteomic analyses has been well demonstrated by many over the past decade. The vast majority of reported approaches has focused primarily on improving the separation once the sample has already been digested. The work presented in this study shows an improvement in multidimensional approaches by prefractionation of intact proteins prior to digestion and separation of the peptides. Two modes of intact protein separation were compared, anion-exchange and reversed-phase, to assess the utility of each mode for the purpose of fractionation. Each of the samples was then enzymatically digested and analyzed by RP-UPLC-MS(E). To assess the validity of each approach, baker's yeast (Saccharomyces cerevisiae) was grown on two different carbon sources, glycerol and dextrose. More proteins were identified by the reversed-phase prefractionation approach (546) than were found by the anion-exchange method (262). As a result, there was much greater coverage of the metabolic pathways of interest for the reversed-phase method than for the anion-exchange method.

Neuroproteomic profiling of human brain tissue using multidimensional separation techniques and selective enrichment of membrane proteins

Hydrophobic membrane proteins (MPs) occupy a unique niche in the brain proteome research due to their important physiological roles. Therefore, the extraction, separation, and identification of MPs are of great interest in proteomic analysis. We applied various proteomic techniques to enrich, separate, and analyze the human brain proteome, including membrane proteome. Temperature-induced phase fractionation with the nonionic surfactant Triton X-114 was used to simultaneously extract, separate, and concentrate low abundant hydrophobic and high abundant hydrophilic proteins from human brain tissue. The extracted and delipidated proteins were analyzed by two-dimensional gel electrophoresis (2DE). Approximately 600 spots were detected in the gels. In-solution digestion was performed on 3 kDa spin filters. Tryptic peptides were separated using RP nano-LC and analyzed using two different high performance mass spectrometers, linear ion trap-Fourier transform and a linear ion trap-Orbitrap to reveal the low abundant MPs. In total, 837 and 780 unique proteins were identified by using linear ion trap-Fourier transform and linear ion trap-Orbitrap mass spectrometers, respectively. More than 29% of the identified proteins were classified as MPs with significant biological functions such as ion channels and transporters. Our study establishes a simple and rapid shotgun approach for the characterization of the brain proteome, and allows comprehensive analysis of brain membrane proteomes.

Macroporous polymer monoliths as second dimension columns in comprehensive two-dimensional gas chromatography: A feasibility study

When the typical column combinations are used, comprehensive two-dimensional gas chromatography (GC×GC) suffers from the impossibility to operate both dimensions at their optimum carrier gas velocities at the same time. This as a result of the flow mismatch caused by the different dimensions of the columns used. The objective of the present study was the development of monolithic second dimension columns which would allow simultaneous optimum-velocity operation. With monolithic GC columns the optimum performance can be obtained at any given flow rate by varying the bed structure and column diameter. Different divinylbenzene-based monolithic columns were prepared and evaluated in terms of permeability and performance. Plate heights of less than 0.18mm and plate generation rates up to 600 plates/s were achieved. 1D-GC experiments performed on short monolithic columns showed a good resolving power thanks to the elevated retention and the good selectivity. A peak capacity up to 12 peaks per 4–5s was obtained for low-boiling alkanes, confirming the potential for fast separations. Excellent repeatability in terms of retention times (RSD<0.5%) and peak widths (RSD<1.5%) was observed. The columns prepared were successfully used in the second dimension of a GC×GC setup with a standard non-polar first dimension. Model experiments proved the possibility to operate both dimensions at their optimum linear velocity simultaneously. The suitability of the novel second dimension column format to perform multidimensional separations was shown for selected applications.

CNTF receptor subunit α as a marker for glioma tumor-initiating cells and tumor grade

Tumor-initiating cells are uniquely resilient to current treatment modalities and play an important role in tumor resistance and recurrence. The lack of specific tumor-initiating cell markers to identify and target these cells presents a major obstacle to effective directed therapy. To identify tumor-initiating cell markers in primary brain tumors, the authors compared the proteomes of glioma tumor-initiating cells to their differentiated progeny using a novel, nongel/shotgun-based, multidimensional liquid-chromatography protein separation technique. An in vivo xenograft model was used to demonstrate the tumorigenic and stem cell properties of these cells. Western blot and immunofluorescence analyses were used to confirm findings of upregulated ciliary neurotrophic factor receptor subunit-α (CNTFRα) in undifferentiated tumor-initiating cells and gliomas of increasing tumor grade. Sequencing of the CNTFRα coding regions was performed for mutation analysis. Finally, antibody-dependent cell-mediated cytotoxicity was used to establish the role of CNTFRα as a potential immunotherapeutic target. Ciliary neurotrophic factor receptor subunit-α expression was increased in tumor-initiating cells and was decreased in the cells' differentiated progeny, and expression levels increased with glioma grade. Mutations of CNTFRα are not common in gliomas. Functional studies using CNTF treatment in glioma tumor-initiating cells showed induction of differentiation through the CNTFRα pathway. Treatment with anti-CNTFRα antibody resulted in increased antibody-dependent cell-mediated cytotoxicity in CNTFRα expressing DAOY cells but not in cell lines that lack CNTFRα. These data indicate that CNTFRα plays a role in the formation or maintenance of tumor-initiating cells in gliomas, is a marker that correlates with histological grade, may underlie treatment resistance in some cases, and is a potential therapeutic target.

Microfluidic Pressure Driven Liquid Chromatography of Biologically Relevant Samples

An overview of the literature regarding the most recent and innovative developments in microfluidic devices for pressure-driven chromatographic separations is given, with a focus on proteomics and metabolomics applications. The applications can be considered as the main driving force for the developments in this research field, since they put high demands on the analytical technology such as for throughput, efficiency, and sensitivity and for the possibilities to interface with mass spectrometry. The developments are evaluated based on the feasibility for use in work flows for the analysis of biologically relevant samples. The literature up to the first half of 2011 is covered. Electrophoretic separations are not within the scope of this review. Several strategies have been described to obtain a retentive phase in microfluidic channels. Open channels with the stationary phase bound to the walls appear to be relatively easy to make. However, the retention in such channels is generally very low for separations of relevant samples. Microfabrication of perfectly ordered topographic structures is the most innovative of the methods discussed for the creation of stationary phases in narrow channels. Several groups work on the improvement of the surface-to-volume ratio in such channels, using different methods, and the developments towards real applications are promising. Channels packed with spherical particles and in situ polymerized monoliths for pressure-driven separations are the most frequently applied. Microfluidic devices with an integrated injection system, a (packed) separation column and a spray tip for coupling to a mass spectrometer are already commercially available, and used in practice in proteomics and metabolomics. Finally, the inherent advantages of microfluidic devices for multidimensional separations have been shown in practice in a number of studies. In these studies, pressure-driven chromatography is coupled (in series or multiplexed) to an electrophoretic separation method. The high peak capacity of such 2-dimensional separations has been shown.

Limits of Detection and Quantification in Comprehensive Multidimensional Separations. 1. A Theoretical Look

Comprehensive multidimensional separations (e.g., GC×GC, LC×LC, etc.) are increasingly popular tools for the analysis of complex samples, due to their many advantages, such as vastly increased peak capacity, and improvements in sensitivity. The most well-established of these techniques, GC×GC, has revolutionized analytical separations in fields as diverse as petroleum, environmental research, food and flavors, and metabolic profiling. Using multidimensional approaches, analytes can be quantified at levels substantially lower than those possible by one-dimensional techniques. However, it has also been shown that the modulation process introduces a new source of error to the measurement. In this work, we present the results of a study into the limits of quantification and detection (LOQ and LOD) in comprehensive multidimensional separations using GC×GC and the more popular "two-step" integration algorithm as an example. Simulation of chromatographic data permits precise control of relevant parameters of peak geometry and modulation phase. Results are expressed in terms of the dimensionless parameter of signal-to-noise ratio of the base peak (S/N(BP)) making them transportable to any result where quantification is performed using a two-step algorithm. Based on these results, the LOD is found to depend upon the modulation ratio used for the experiment and vary between a S/N(BP) of 10-17, while the LOQ depends on both the modulation ratio and the phase of the modulation for the peak and ranges from a S/N(BP) of 10 to 50, depending on the circumstances.

Combining multidimensional liquid chromatography and MALDI–TOF-MS for the fingerprint analysis of secreted peptides from the unexplored sea anemone species Phymanthus crucifer

Sea anemones are sources of biologically active proteins and peptides. However, up to date few peptidomic studies of these organisms are known; therefore most species and their peptide diversity remain unexplored. Contrasting to previous venom peptidomic works on sea anemones and other venomous animals, in the present study we combined pH gradient ion-exchange chromatography with gel filtration and reversed-phase chromatography, allowing the separation of the 1–10kDa polypeptides from the secretion of the unexplored sea anemone Phymanthus crucifer (Cnidaria/Phymanthidae). This multidimensional chromatographic approach followed by MALDI–TOF-MS detection generated a peptide fingerprint comprising 504 different molecular mass values from acidic and basic peptides, being the largest number estimated for a sea anemone exudate. The peptide population within the 2.0–3.5kDa mass range showed the highest frequency whereas the main biomarkers comprised acidic and basic peptides with molecular masses within 2.5–6.9kDa, in contrast to the homogeneous group of 4–5kDa biomarkers found in sea anemones such as B. granulifera and B. cangicum (Cnidaria/Actiniidae). Our study shows that sea anemone peptide fingerprinting can be greatly improved by including pH gradient ion-exchange chromatography into the multidimensional separation approach, complemented by MALDI–TOF-MS detection. This strategy allowed us to find the most abundant and unprecedented diversity of secreted components from a sea anemone exudate, indicating that the search for novel biologically active peptides from these organisms has much greater potential than previously predicted.

Neuropeptide analysis with liquid chromatography‐capillary electrophoresis‐mass spectrometric imaging

Herein we report the first attempt of coupling multidimensional separations to matrix-assisted laser desorption/ionization (MALDI) mass spectrometric imaging detection. Complex neuropeptide mixtures extracted from crustaceans were first fractionated by reversed-phase liquid chromatography (RPLC), and then subjected to a capillary electrophoresis-mass spectrometric imaging platform. With a specific focus on orcokinin family neuropeptides, we demonstrated that these trace-level analytes from complex neural tissue samples can be fully separated from chemical noise and interfering components and visualized as mass spectrometric imaging signals. A total of 19 putative orcokinins were detected, with highly efficient separations within the family being achieved for the first time. The results indicate that two-dimensional separation coupling to mass spectrometric imaging can serve as a novel and powerful tool in proteomics and peptidomics studies.

Handling and analysis of cells and bioparticles on centrifugal microfluidic platforms

Microfluidic systems for cell separation and analysis have attracted increasing research activity over the past decades. In particular, the prospect of integrating all steps from sample preparation to assay readout in a single microfluidic cartridge, which is inserted into a compact, portable and potentially low-cost instrument, bears great promise to leverage next-generation diagnostic products and to advance life-science research with novel cell and particle manipulation, and analysis tools. Within the range of microfluidic actuation principles available, the centrifugal force is exceptionally well suited for cell handling due to its rotationally induced ‘artificial gravity field’, which can be varied over several orders of magnitude and which can manipulate bioparticles even in the absence of flow. We will survey how the base centrifugal force has been combined with the hydrodynamic Stokes drag, magnetic, dielectrophoretic and other forces to enable multidimensional separation and manipulation. The same centrifugal microfluidic toolbox has also been applied to investigate particles such as biofunctionalized beads, bacteria and multicellular microorganisms. This review summarizes the significant progress in modular unit operations such as cell removal, filtering, lysis, separation, sorting, encapsulation, trapping, assaying, sensing, cytometry and detection, even derived from low-cost conventional optical disc drive technology (e.g., CD and DVD), towards integrated and automated centrifugal microfluidic platforms for the handling and analysis of cells and bioparticles.

Critical Comparison of Multidimensional Separation Methods for Increasing Protein Expression Coverage

We present a comparison of two-dimensional separation methods and how they affect the degree of coverage of protein expression in complex mixtures. We investigated the relative merits of various protein and peptide separations prior to acidic reversed-phase chromatography directly coupled to an ion trap mass spectrometer. The first dimensions investigated were density gradient organelle fractionation of cell extracts, 1D SDS-PAGE protein separation followed by digestion by trypsin or GluC proteases, strong cation exchange chromatography, and off-gel isoelectric focusing of tryptic peptides. The number of fractions from each first dimension and the total data accumulation RP-HPLC-MS/MS time was kept constant and the experiments were run in triplicate. We find that the most critical parameters are the data accumulation time, which defines the level of under-sampling and the avoidance of peptides from high expression level proteins eluting over the entire gradient.

A Review of Chemometrics Applied to Comprehensive Two-dimensional Separations from 2008–2010

This review article covers developments in multidimensional separations combined with chemometrics that were published in 2008 through 2010, specifically for multidimensional gas chromatography, liquid chromatography, and electrophoresis. Although different instrumentation is used to generate multidimensional separations data, many similar data processing options and chemometrics can be applied in order to objectively distill the data into useful knowledge while reducing manual analysis and preserving data integrity. This review article describes the chemometrics employed in the referenced studies in terms of unsupervised, supervised, preprocessing, resolution, and image analysis algorithms. Other factors that affect converting data into useful knowledge are the structure of the data and the format of the data submitted to the analysis methods, so the studies are also described in terms of data dimensionality and data format (i.e., whether peak tables or raw data points were analyzed).

Optimal selection of 2D reversed-phase–reversed-phase HPLC separation techniques in bottom-up proteomics

Evaluation of: Stephanowitz H, Lange S, Lang D et al. Improved two-dimensional reversed phase–reversed phase LC-MS/MS approach for identification of peptide–protein interactions. J. Proteome Res. 11(2), 1175–1183 (2011).Recent developments in bottom-up proteomics have supplanted the use of gel-based approaches in favor of multidimensional chromatographic separations of peptide mixtures followed by mass spectrometry analysis. This trend is driven by the desire to eliminate labor-intensive in-gel digestion procedures and increase proteome coverage through better recovery of proteolytic fragments. Introduction of reversed-phase–reversed-phase 2D separation techniques is one of the major improvements that have made this possible. In this article, we review recent developments in 2D HPLC and highlight variations in reversed-phase HPLC separation selectivity that allow for superior peak capacity in peptide fractionation.

Quantitative Proteomic Analysis of Mouse Embryonic Fibroblasts and Induced Pluripotent Stem Cells Using 16O/18O Labeling

Induced pluripotent stem cells (iPSC) hold great promise for regenerative medicine as well as for investigations into the pathogenesis and treatment of various diseases. Understanding of key intracellular signaling pathways and protein targets that control development of iPSC from somatic cells is essential for designing new approaches to improve reprogramming efficiency. Here, we report the development and application of an integrated quantitative proteomics platform for investigating differences in protein expressions between mouse embryonic fibroblasts (MEF) and MEF-derived iPSC. This platform consists of 16O/18O labeling, multidimensional peptide separation coupled with tandem mass spectrometry, and data analysis with UNiquant software. With this platform, a total of 2481 proteins were identified and quantified from the 16O/18O-labeled MEF-iPSC proteome mixtures with a false discovery rate of 0.01. Among them, 218 proteins were significantly upregulated, while 247 proteins were significantly downregulated in iPSC compared to MEF. Many nuclear proteins, including Hdac1, Dnmt1, Pcna, Ccnd1, Smarcc1, and subunits in DNA replication and RNA polymerase II complex, were found to be enhanced in iPSC. Protein network analysis revealed that Pcna functions as a hub orchestrating complicated mechanisms including DNA replication, epigenetic inheritance (Dnmt1), and chromatin remodeling (Smarcc1) to reprogram MEF and maintain stemness of iPSC.

Multidimensional plasma protein separation technique for identification of potential Alzheimer’s disease plasma biomarkers: a pilot study

Neurochemical differential diagnosis between Alzheimer's disease and other dementias is currently based on CSF biomarkers. Here, we report a method by which potential biomarkers can be identified in blood. Blood plasma samples from seven well-characterized Alzheimer's patients and seven non-Alzheimer's patients were subjected to a multi-dimensional protein separation procedure. After removal of 12 high-abundance proteins, the depleted samples from both diagnostic groups were labeled with different fluorescent dyes, mixed and separated by anion exchange and RP-chromatography. The resulting chromatography fractions were analyzed on 2D-gels. Twenty significant differentially expressed proteins were identified by mass spectrometry analysis. Ten of these proteins were either involved in the pathophysiology of Alzheimer's disease or the amyloid/Aβ-peptide processing pathway. This work demonstrated a successful application of a multidimensional separation technique and holds the potential to identifying blood-based biomarkers for other diseases in the future.

A new class of multi-dimensional Teager-Kaiser and higher order operators based on directional derivatives

This work aims at introducing some energy operators linked to Teager-Kaiser energy operator (TKEO) (Kaiser in On a simple algorithm to calculate the energy of a signal, pp 381---384, 1990), its associated higher order versions and expanding them to multi-dimensional signals. These operators are very useful for analysing oscillatory signals with time-varying amplitude and frequency (AM---FM). We first propose a new mathematical expression of these operators using directional derivatives along any n-D vector and Kronecker powers (Proposition 1, Sect. 3). This mathematical formulation allows us to extend to n-D case some properties of the classical TKEO such as tracking of AM envelope and instantaneous frequency of a multi-dimensional AM---FM signal. In addition, we have introduced a new scalar function using the directional derivative along a vector to recover the sign of the frequency components. Applications of this model to a local n-D AM---FM signal and the related demodulation errors are presented. To show the effectiveness and the robustness of the new class of operators in term of envelope and frequency tracking, results obtained on synthetic and real data are compared to multi-dimensional energy separation algorithm (Maragos and Bovik in J Opt Soc Am A 12:1867---1876, 1995) and to our previously developed method (Salzenstein and Boudraa in Signal Process 89(4):623---640, 2009). Finally, the performances of these methods are investigated in the presence of an additive noise.

In-house construction of a UHPLC system enabling the identification of over 4000 protein groups in a single analysis

Here, we describe an in-house built ultra-high pressure liquid chromatography (UHPLC) system, with little complexity in design and high separation power combined with convenience in operation. This system enables the use of long columns of 40 cm packed with 1.8 μm particles generating pressures below 1000 bar. Furthermore, the system could be operated at flow rates between 50 and 200 nL min(-1) while maintaining its separation power. Several gradients were optimized ranging from 23 to 458 minutes. With the longest gradient we identified over 4500 protein groups and more than 26,000 unique peptides from 1 μg of a human cancer cell lysate in a single run using an Orbitrap Velos - a level of performance often seen solely using multidimensional separation strategies. Further experiments using a mass spectrometer with faster sequencing speeds, like the TripleTOF 5600, enabled us to identify over 1400 protein groups in a 23 min gradient. The TripleTOF 5600 performed especially well, compared to the Orbitrap Velos, for the shorter gradients used. Our data demonstrate that the combination of UHPLC with high resolution mass spectrometry at increased sequencing speeds enables extensive proteome analysis in single runs.

대량 발굴 프로테옴 분석을 위한 어쿠스틱 기술 기반 고속 단백질 절편화

최근 질량분석기와 다차원 크로마토그래피 분석법과 대용량 데이터를 처리하는 생물정보학 프로그램 등과 같은 복합적인 기술요소들의 발전은 대량 프로테옴을 분석하는 것을 가능하게 했다. 하지만, 이런 대량의 프로테옴을 분석하는 것은 단백질 절편화 과정의 긴 소요 시간과 낮은 재현성으로 인하여 한계를 가진다. 이 연구에서는 어쿠스틱 기술을 이용해 빠르게 단백질을 분해하는 새로운 방법을 제시했다. 이 어쿠스틱 기술의 시간과 강도를 최적화하기 위해서 여러 가지 조건에서 BSA 단백질을 트립신으로 절편화한 후 액체 크로마토그래피와 질량분석기로 분석하였다. 16시간동안 인큐베이션하는 기존의 방법과 슈퍼 어쿠스틱 기술을 사용한 방법을 비교하였을 때 슈퍼 어쿠스틱 기술이 기존의 방법보다 더 높은 아미노산 동정율을 보였으며, 유방암 세포와 같은 단백질 복합체에 적용하였을 때 30분의 절편화 시간에서도 효율적인 결과를 확인할 수 있었다. 이 새로운 방법은 샘플 전처리 과정에서 많은 양의 단백질일지라도 더 좋은 효율성을 가지게 되고 또한, 소비되는 시간도 줄여주며 일정 시간내의 샘플 처리량도 증가하게 된다. Recent developments and improvements of multiple technological elements including mass spectrometry (MS) instrument, multi-dimensional chromatographic separation, and software tools processing MS data resulted in benefits of large scale proteomics analysis. However, its throughput is limited by the speed and reproducibility of the protein digestion process. In this study, we demonstrated a new method for rapid proteolytic digestion of proteins using acoustic technology. Tryptic digests of BSA prepared at various conditions by super acoustic for optimization time and intensity were analyzed by LC-MS/MS showed higher sequence coverage in compared with traditional 16 hrs digestion method. The method was applied successfully for complex proteins of a breast cancer cells at 30 min of digestion at intensity 2. This new application reduces time-consuming of sample preparation with better efficiency, even with large amount of proteins, and increases high-throughput process in sample preparation state.