Fractionation Of Intact Proteins Research Articles

Proteomic Profiling of the Tumor Microenvironment.

The tumor microenvironment forms a complex pro-tumorigenic milieu constituted by extracellular matrix, surrounding stroma, infiltrating cell populations, and signaling molecules. Proteomic studies have the potential to reveal how individual cell populations within the tumor tissue modulate the microenvironment through protein secretion and consequently alter their protein expression and localization to adapt to this milieu. As a result, proteomic approaches have uncovered how these dynamic components communicate and promote tumor development and progression. The characterization of these mechanisms is relevant for the identification of clinically targetable pathways and for the development of diagnostic tools. Here we describe a method based on the isolation of individual cell compartments and the chromatographic fractionation of intact proteins, followed by enzymatic digestion of individual fractions, and mass-spectrometry analysis, for the profiling of tumor microenvironment cell populations.

A robust and effective intact protein fractionation strategy by GO/PEI/Au/PEG nanocomposites for human plasma proteome analysis

Identification of human plasma proteins with deep coverage is considered as a great challenge due to its extreme complexity. In this work, an intact proteins fractionation strategy based on multi-interaction between proteins and GO/PEI/Au/PEG nanocomposites (GPAP strategy) was developed for human plasma proteome deep analysis. Compared with untreated method, the number of identified proteins was increased from 858 to 2023, among which the number of low-abundance proteins (< 100ng/mL) was increased from 2 to 11. The concentration range of the identified proteins was broaden to 9 orders of magnitude. Furthermore, the spectral count of the top three proteins in human plasma (Plasma Albumin, Human IgG, Serotransferrin) were decreased in the range of 37.4–82.6%. An excellent reproducibility of GPAP strategy was verified via stable isotope dimethyl label strategy. The functionalized material was demonstrated to be an efficient method to achieve deep coverage identification of human plasma proteome.

Top-Down Proteomics of Large Proteins up to 223 kDa Enabled by Serial Size Exclusion Chromatography Strategy.

Mass spectrometry (MS)-based top-down proteomics is a powerful method for the comprehensive analysis of proteoforms that arise from genetic variations and post-translational modifications (PTMs). However, top-down MS analysis of high molecular weight (MW) proteins remains challenging mainly due to the exponential decay of signal-to-noise ratio with increasing MW. Size exclusion chromatography (SEC) is a favored method for size-based separation of biomacromolecules but typically suffers from low resolution. Herein, we developed a serial size exclusion chromatography (sSEC) strategy to enable high-resolution size-based fractionation of intact proteins (10-223 kDa) from complex protein mixtures. The sSEC fractions could be further separated by reverse phase chromatography (RPC) coupled online with high-resolution MS. We have shown that two-dimensional (2D) sSEC-RPC allowed for the identification of 4044 more unique proteoforms and a 15-fold increase in the detection of proteins above 60 kDa, compared to one-dimensional (1D) RPC. Notably, effective sSEC-RPC separation of proteins significantly enhanced the detection of high MW proteins up to 223 kDa and also revealed low abundance proteoforms that are post-translationally modified. This sSEC method is MS-friendly, robust, and reproducible and, thus, can be applied to both high-efficiency protein purification and large-scale proteomics analysis of cell or tissue lysate for enhanced proteome coverage, particularly for low abundance and high MW proteoforms.

Multi-Lectin Affinity Chromatography for Separation, Identification, and Quantitation of Intact Protein Glycoforms in Complex Biological Mixtures.

Protein glycosylation is considered to be one of the most abundant post-translational modifications and is recognized for playing key roles in cellular functions. Aberrant N-linked glycosylation has been associated with several human diseases and has prompted the development and constant improvement of analytical tools to separate, characterize, and quantify glycoproteins in complex mixtures extracted from various biological samples (such as blood and tissue). Lectins, or carbohydrate-binding proteins, have been used as valuable tools for enriching for glycoproteins and selecting for specific types of glycosylation. Herein a method using multidimensional intact protein fractionation and LC-MS/MS analysis is described. Immunodepletion is used to remove highly abundant proteins from human plasma, followed by glycoform separation using multi-lectin affinity chromatography, in which specific lectins are chosen to capture and elute specific types of glycosylation. Reversed-phase chromatography prior to digestion is used for further fractionation, allowing for an increased number of protein identifications of moderate- to low-abundant proteins detectable in plasma. This method also incorporates isotopic labeling during alkylation for relative quantitation between two samples (such as a case and control). A bottom-up, tandem mass spectrometry-based proteomics approach is used for protein identification and quantitation, and allows for screening glycoform-specific changes across hundreds of plasma proteins.

Mass spectrometry based proteomics for absolute quantification of proteins from tumor cells

In-depth quantitative profiling of the proteome and sub-proteomes of tumor cells has relevance to tumor classification, the development of novel therapeutics, and of prognostic and predictive markers and to disease monitoring. In particular the tumor cell surface represents a highly relevant compartment for the development of targeted therapeutics and immunotherapy. We have developed a proteomic platform to profile tumor cells that encompasses enrichment of surface membrane proteins, intact protein fractionation and label-free mass spectrometry based absolute quantification. Here we describe the methodology for capture, identification and quantification of cell surface proteins using biotinylation for labeling of the cell surface, avidin for capture of biotinylated proteins and ion mobility mass spectrometry for protein identification and quantification.

Top Down Proteomics

The field of proteomics has reached a level of maturity where significant biological discoveries are being made with current technology. By far the largest numbers of proteomics experiments employ a “bottom-up” protein analysis. In this strategy proteins or protein mixtures are digested with a protease and then analyzed en masse in a “shotgun” format. Tandem mass spectrometry is then used to sequence peptides, and informatics tools are used to identify the sequences and assign them to proteins or genes. This same strategy can be used to identify modifications to peptides. This strategy works very well to identify the proteins present and their modifications. A challenge for bottom-up proteomics is the proper assignment of protein isoforms and patterns of modifications that create the many proteoforms present in cells. Why is this important? The functional sophistication of complex organisms, like humans, is driven by the ability to use a limited number of gene sequences in many different ways. Determining the precise proteoform involved in a specific function will be key for dissecting out the molecular mechanisms used by eukaryotic organisms. A key technology to determine this information is “top down” mass spectrometry. In this method, intact proteins are analyzed to both identify the protein and to determine the identity and location of modifications within the protein using a tandem mass spectrometer. The analysis of intact proteins by mass spectrometry is a major challenge. The first problem for top down analysis is fractionation of proteins. Traditional methods such as gel electrophoresis have limitations on the recovery of proteins in a form suitable for analysis by mass spectrometry. Extracting gel-separated proteins from the polyacrylamide matrix requires electroelution or electroblotting, frequently resulting in sample losses. Chromatographic methods to separate intact proteins can work well, but the resolving power for intact proteins with more than ~500 amino acids is poor. After introduction into the mass spectrometer, amide bonds in the protein backbone must be fragmented to create ladders of sequence information. Because proteins have many chemical bonds that can “soak up” energy put into ions, vigorous research into excitation methods to more efficiently and completely fragment intact protein ions is bearing fruit. The nonergodic methods for protein ion excitation are increasingly able to fragment larger and larger protein ions, which when coupled with improvements in mass resolving power are aiding in robust protein identification. A key technological change underway is the development of lower cost mass spectrometers that are able to analyze intact proteins. This change will put the technology into more hands, which will speed up innovations and developments. A recent American Society for Mass Spectrometry Sanibel Conference meeting for top down mass spectrometry was very well attended and the lectures demonstrated a vibrant and growing subfield of mass spectrometry-based proteomics and biopharmaceutical analysis. To complement this very successful meeting and highlight a growth area, the Journal of Proteome Research and Analytical Chemistry have assembled and published a joint thematic virtual issue of the papers published in these journals on top down proteomics. This thematic issue emphasizes the challenges and successes in the field from preparation and fractionation of intact proteins to methods for direct fragmentation of whole proteins and the informatics for their identification by database retrieval. Recent developments clearly show that top down proteomics is a vigorous and energized area of research as described in a recent Chemical and Engineering News article by Celia Arnaud.

Coupling a Detergent Lysis/Cleanup Methodology with Intact Protein Fractionation for Enhanced Proteome Characterization

The expanding use of surfactants for proteome sample preparations has prompted the need to systematically optimize the application and removal of these MS-deleterious agents prior to proteome measurements. Here we compare four detergent cleanup methods (trichloroacetic acid (TCA) precipitation, chloroform/methanol/water (CMW) extraction, a commercial detergent removal spin column method (DRS) and filter-aided sample preparation (FASP)) to provide efficiency benchmarks with respect to protein, peptide, and spectral identifications in each case. Our results show that for protein-limited samples, FASP outperforms the other three cleanup methods, while at high protein amounts, all the methods are comparable. This information was used to investigate and contrast molecular weight-based fractionated with unfractionated lysates from three increasingly complex samples ( Escherichia coli K-12, a five microbial isolate mixture, and a natural microbial community groundwater sample), all of which were prepared with an SDS-FASP approach. The additional fractionation step enhanced the number of protein identifications by 8% to 25% over the unfractionated approach across the three samples.

Mapping intact protein isoforms in discovery mode using top-down proteomics

A full description of the human proteome relies on the challenging task of detecting mature and changing forms of protein molecules in the body. Large scale proteome analysis1 has routinely involved digesting intact proteins followed by inferred protein identification using mass spectrometry (MS)2. This “bottom up” process affords a high number of identifications (not always unique to a single gene). However, complications arise from incomplete or ambiguous2 characterization of alternative splice forms, diverse modifications (e.g., acetylation and methylation), and endogenous protein cleavages, especially when combinations of these create complex patterns of intact protein isoforms and species3. “Top down” interrogation of whole proteins can overcome these problems for individual proteins4,5, but has not been achieved on a proteome scale due to the lack of intact protein fractionation methods that are well integrated with tandem MS. Here we show, using a new four dimensional (4D) separation system, identification of 1,043 gene products from human cells that are dispersed into >3,000 protein species created by post-translational modification, RNA splicing, and proteolysis. The overall system produced >20-fold increases in both separation power and proteome coverage, enabling the identification of proteins up to 105 kilodaltons and those with up to 11 transmembrane helices. Many previously undetected isoforms of endogenous human proteins were mapped, including changes in multiply-modified species in response to accelerated cellular aging (senescence) induced by DNA damage. Integrated with the latest version of the Swiss-Prot database6, the data provide precise correlations to individual genes and proof-of-concept for large scale interrogation of whole protein molecules. The technology promises to improve the link between proteomics data and complex phenotypes in basic biology and disease research7.

Integrated mass spectrometry–based analysis of plasma glycoproteins and their glycan modifications

We present a protocol for the identification of glycosylated proteins in plasma followed by elucidation of their individual glycan compositions. The study of glycoproteins by mass spectrometry is usually based on cleavage of glycans followed by separate analysis of glycans and deglycosylated proteins, which limits the ability to derive glycan compositions for individual glycoproteins. The methodology described here consists of 2D HPLC fractionation of intact proteins and liquid chromatography-multistage tandem mass spectrometry (LC-MS/MS(n)) analysis of digested protein fractions. Protein samples are separated by 1D anion-exchange chromatography (AEX) with an eight-step salt elution. Protein fractions from each of the eight AEX elution steps are transferred onto the 2D reversed-phase column to further separate proteins. A digital ion trap mass spectrometer with a wide mass range is then used for LC-MS/MS(n) analysis of intact glycopeptides from the 2D HPLC fractions. Both peptide and oligosaccharide compositions are revealed by analysis of the ion fragmentation patterns of glycopeptides with an intact glycopeptide analysis pipeline.

Dynamics of Protein Damage in Yeast Frataxin Mutant Exposed to Oxidative Stress

Oxidative stress and protein carbonylation is implicated in aging and various diseases such as neurodegenerative disorders, diabetes, and cancer. Therefore, the accurate identification and quantification of protein carbonylation may lead to the discovery of new biomarkers. We have developed a new method that combines avidin affinity selection of carbonylated proteins with iTRAQ labeling and LC fractionation of intact proteins. This simple LC-based workflow is an effective technique to reduce sample complexity, minimize technical variation, and enable simultaneous quantification of four samples. This method was used to determine protein oxidation in an iron accumulating mutant of Saccharomyces cerevisiae exposed to oxidative stress. Overall, 31 proteins were identified with 99% peptide confidence, and of those, 27 proteins were quantified. Most of the identified proteins were associated with energy metabolism (32.3%), and cellular defense, transport, and folding (38.7%), suggesting a drop in energy production and reducing power of the cells due to the damage of glycolytic enzymes and decrease in activity of enzymes involved in protein protection and regeneration. In addition, the oxidation sites of seven proteins were identified and their estimated position also indicated a potential impact on the enzymatic activities. Predicted 3D structures of peroxiredoxin (TSA1) and thioredoxin II (TRX2) revealed close proximity of all oxidized amino acid residues to the protein active sites.

Protein- versus peptide fractionation in the first dimension of two-dimensional high-performance liquid chromatography-matrix-assisted laser desorption/ionization tandem mass spectrometry for qualitative proteome analysis of tissue samples

The availability of robust and highly efficient separation methods represents a major requirement for proteome analysis. This study investigated the characteristics of two different gel-free proteomic approaches to the fractionation of proteolytic peptides and intact proteins, respectively, in a first separation dimension. Separation and mass spectrometric detection by matrix-assisted laser desorption/ionization tandem mass spectrometry (MALDI-MS/MS) were performed at the peptide level in both methods. Bottom-up analysis (BU) was carried out employing well established peptide fractionation in the first separation dimension by strong cation-exchange chromatography (SCX), followed by ion-pair reversed-phase chromatography (IP-RPC) in the second dimension. In the semi-top-down approach (STD), which involved intact protein fractionation in the first dimension, the separation mode in both dimensions was IP-RPC utilizing monolithic columns. Application of the two approaches to the proteome analysis of proteins extracted from a tumor tissue revealed that the BU method identified more proteins (1245 in BU versus 920 in STD) while STD analysis offered higher sequence coverage (14.8% in BU versus 17.5% in STD on average). The identification of more basic and larger proteins was slightly favored in the BU approach, most probably due to higher losses of these proteins during intact protein handling and separation in the STD method. A significant degree of complementarity was revealed by an approximately 33% overlap between one BU and STD replicate, while 33% each of the protein identifications were unique to both methods. In the STD method, peptides obtained upon digestion of the proteins contained in fractions of the first separation dimension covered a broad elution window in the second-dimension separation, which demonstrates a high degree of “pseudo-orthogonality” of protein and peptide separation by IP-RPC in both separation dimensions.

Proteomic Study of Human Glioblastoma Multiforme Tissue Employing Complementary Two-Dimensional Liquid Chromatography- and Mass Spectrometry-Based Approaches

An extensive data set comprising 2660 unique protein identifications was obtained for the proteome of a human brain tumor (glioblastoma multiforme) by combining the results of two complementary analytical strategies based on two-dimensional chromatography and mass spectrometry. A bottom-up method, performing peptide separation in both chromatographic dimensions was employed as well as a semi-top-down method, in which intact proteins were separated in the first and tryptic peptides in the second dimension. The identified proteins were assigned to their molecular functions and compared to previously identified proteins of glioblastoma multiforme (= astrocytoma WHO grade IV), lower WHO grade astrocytomas (grade II and III), and nontumor brain tissue. With the use of a subset of 104 identified membrane proteins, the properties of intact protein fractionation in the first dimension of the semi-top-down approach were elucidated in detail. The benefit of the semi-top-down approach was further demonstrated by the identification of a set of endogenous glioblastoma multiforme expressed proteins. These proteins correspond to recombinant antigens which were recently found to be reactive against autoantibodies in glioblastoma multiforme patients. The results indicate the usefulness of the semi-top-down approach for the investigation of immunogenic antigens in human tumor tissue samples.

A Robust Two-Dimensional Separation for Top-Down Tandem Mass Spectrometry of the Low-Mass Proteome

For fractionation of intact proteins by molecular weight (MW), a sharply improved two-dimensional (2D) separation is presented to drive reproducible and robust fractionation before top-down mass spectrometry of complex mixtures. The "GELFrEE" (i.e., gel-eluted liquid fraction entrapment electrophoresis) approach is implemented by use of Tris-glycine and Tris-tricine gel systems applied to human cytosolic and nuclear extracts from HeLa S3 cells, to achieve a MW-based fractionation of proteins from 5 to >100 kDa in 1 h. For top-down tandem mass spectroscopy (MS/MS) of the low-mass proteome (5-25 kDa), between 5 and 8 gel-elution (GE) fractions are sampled by nanocapillary-LC-MS/MS with 12 or 14.5 tesla Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometers. Single injections give about 40 detectable proteins, about half of which yield automated ProSight identifications. Reproducibility metrics of the system are presented, along with comparative analysis of protein targets in mitotic versus asynchronous cells. We forward this basic 2D approach to facilitate wider implementation of top-down mass spectrometry and a variety of other protein separation and/or characterization approaches.

1 mm ID poly(styrene‐co‐divinylbenzene) monolithic columns for high‐peak capacity one‐ and two‐dimensional liquid chromatographic separations of intact proteins

The LC performance of a 1x50 mm polymer monolithic column format was demonstrated with high-peak capacity one- (1D) and offline two dimensional (2D) LC separations of intact proteins. After optimizing the RP 1D-LC conditions, including column temperature, flow rate and gradient time, a peak capacity of 475 was achieved within a 2-h analysis. The suitability of the monolithic column was also demonstrated for fast 1 min protein separations yielding 1 s peak widths determined at half peak height. In addition, an offline 2D-LC method was developed using the micro-fraction collection capabilities of the autosampler allowing automatic fractionation of intact proteins after the weak-ion-exchange (WAX) separation, and re-injection of the fractions onto the second-dimension RP monolithic column. The best peak capacity-to-analysis time ratio was obtained when applying 10 min second-dimension RP gradients. At optimized conditions, the WAX/x/RPLC separation of intact Escherichia coli proteins was performed within 6 h yielding a maximum theoretical peak capacity of 4880.

Subcellular protein extraction from human pancreatic cancer tissues

Proteins are the major class of effector molecules in cellular systems. For the identification of functional differences between normal and diseased tissues, a reliable analysis of their protein content is essential. Reproducible isolation and fractionation of intact proteins are important in this respect, but their complexity in structure and concentration, their close interaction, and their instability represent major challenges. For protein isolation in tissues, the breakdown of cell-cell and cell-matrix connections within a tissue without affecting protein quality is a critical factor. We compared different processes for a compartmental protein preparation from pancreatic tissue, one of the most challenging tissues for protein isolation because of its high protease content. Success of the different procedures varied greatly. Based on a scheme of tissue-slicing and subsequent cell isolation, we established a reliable workflow for the fractional extraction of cytosolic proteins, membrane and organelle proteins, nuclear proteins, and cytoskeletal filaments. The tissue slices also allow for a representative confirmation of individual samples' cellular status by histochemical processes, and a proper separation or mixing of cellular material from across a tumor if required.

Comparative analysis of the liver and plasma proteomes as a novel and powerful strategy for hepatocellular carcinoma biomarker discovery

The extraordinary developments made in the past decade in proteomic technologies, in particular in mass spectrometry, have enabled investigators to consider designing studies to search for diagnostic and therapeutic biomarkers by scanning complex proteome samples. We developed a method based on extensive fractionation of intact proteins, to comprehensively and quantitatively profile the liver and plasma proteomes in health and disease. We have applied this method to samples collected from patients with early hepatocellular carcinoma (HCC) and from patients with liver cirrhosis as well as to samples collected from three mouse models of HCC. This method allowed for the identification of proteins that differ in expression levels in liver tissue or in plasma with disease progression from liver fibrosis, cirrhosis or steatohepatitis to HCC. The comparative analysis of the liver and plasma proteomes generated from human and mouse specimens, constitutes a novel and powerful strategy for HCC biomarker discovery.

Comprehensive and quantitative proteome profiling of the mouse liver and plasma

We report a comprehensive and quantitative analysis of the mouse liver and plasma proteomes. The method used is based on extensive fractionation of intact proteins, further separation of proteins based on their abundance and size, and high-accuracy mass spectrometry. This analysis reached a depth in proteomic profiling not reported to date for a mammalian tissue or a biological fluid, with 7099 and 4727 proteins identified with high confidence in the liver and in the corresponding plasma, respectively. This method allowed for the identification in both compartments of low-abundance proteins such as cytokines, chemokines, and receptors and for the detection in plasma of proteins in the pg/mL concentration range. This method also allowed for semiquantitation of all identified proteins. The calculated abundance scores correlated with the abundance of the corresponding transcripts for the large majority of the proteins identified in the liver. Finally, comparison of the liver and plasma datasets demonstrated that a significant number of proteins identified in the liver can be detected in plasma. These included proteins involved in complement and coagulation, in fatty acid, purine and pyruvate metabolism, in gluconeogenesis and glycolysis, in protein ubiquitination, and in insulin, interleukin-4, epidermal growth factor, and platelet-derived growth factor signaling. This in-depth analysis of the mouse liver and corresponding plasma proteomes provides a strong basis for investigations of liver pathobiology and biology that employ mouse models of hepatic diseases in an effort to better understand, diagnose, treat, and prevent human hepatic diseases.

Proteome Profiling of Breast Tumors by Gel Electrophoresis and Nanoscale Electrospray Ionization Mass Spectrometry

We have conducted proteome-wide analysis of fresh surgery specimens derived from breast cancer patients, using an approach that integrates size-based intact protein fractionation, nanoscale liquid separation of peptides, electrospray ion trap mass spectrometry, and bioinformatics. Through this approach, we have acquired a large amount of peptide fragmentation spectra from size-resolved fractions of the proteomes of several breast tumors, tissue peripheral to the tumor, and samples from patients undergoing noncancer surgery. Label-free quantitation was used to generate protein abundance maps for each proteome and perform comparative analyses. The mass spectrometry data revealed distinct qualitative and quantitative patterns distinguishing the tumors from healthy tissue as well as differences between metastatic and non-metastatic human breast cancers including many established and potential novel candidate protein biomarkers. Selected proteins were evaluated by Western blotting using tumors grouped according to histological grade, size, and receptor expression but differing in nodal status. Immunohistochemical analysis of a wide panel of breast tumors was conducted to assess expression in different types of breast cancers and the cellular distribution of the candidate proteins. These experiments provided further insights and an independent validation of the data obtained by mass spectrometry and revealed the potential of this approach for establishing multimodal markers for early metastasis, therapy outcomes, prognosis, and diagnosis in the future.

Contribution of Protein Fractionation to Depth of Analysis of the Serum and Plasma Proteomes

In-depth analysis of the serum and plasma proteomes by mass spectrometry is challenged by the vast dynamic range of protein abundance and substantial complexity. There is merit in reducing complexity through fractionation to facilitate mass spectrometry analysis of low-abundance proteins. However, fractionation reduces throughput and has the potential of diluting individual proteins or inducing their loss. Here, we have investigated the contribution of extensive fractionation of intact proteins to depth of analysis. Pooled serum depleted of abundant proteins was fractionated by an orthogonal two-dimensional system consisting of anion-exchange and reversed-phase chromatography. The resulting protein fractions were aliquotted; one aliquot was analyzed by shotgun LC-MS/MS, and another was further resolved into protein bands in a third dimension using SDS-PAGE. Individual gel bands were excised and subjected to in situ digestion and mass spectrometry. We demonstrate that increased fractionation results in increased depth of analysis based on total number of proteins identified in serum and based on representation in individual fractions of specific proteins identified in gel bands following a third-dimension SDS gel analysis. An intact protein analysis system (IPAS) based on a two-dimensional plasma fractionation schema was implemented that resulted in identification of 1662 proteins with high confidence with representation of protein isoforms that differed in their chromatographic mobility. Further increase in depth of analysis was accomplished by repeat analysis of aliquots from the same set of two-dimensional fractions resulting in overall identification of 2254 proteins. We conclude that substantial depth of analysis of proteins from milliliter quantities of serum or plasma and detection of isoforms are achieved with depletion of abundant proteins followed by two-dimensional protein fractionation and MS analysis of individual fractions.

A comparison of sample preparation methods for the study of the human serum proteome

This work describes our work of investigating the outcome of different sample preparation methods for label-free quantitative proteomic study of human serum proteins. All analyses were preformed on insolution trypsin digestion of human serum. MS analysis was on a Q-TOF instrument with a nanoflow split-free LC system interfaced to the ion-source (75 μ id LC columns). Data was acquired using data dependent analysis (DDA) or a high/low energy strategy. The high/low method is a “protein expression” method were a mass window of data is acquired in low energy (intact peptide) followed by a high energy acquisition on all ions in the mass window for intact peptides. This maximizes the duty cycle of the Q-TOF to yield extensive qualitative and quantitative information by concurrently analyzing the peptide ions in the mixture. Samples were prepared by a verity of methods including, top-6 or top-20 abundant protein depletion and enhancement (reproducibility) of digestion with trypsin-friendly surfactants. Fractionation of intact proteins by 1D SDS-PAGE and chromatography was investigated. In addition, peptide mixtures from digestion were separated by multidimensional chromatography. The 2D methods included SCX/RP, RP/RP, and HILIC/RP. The objective was to understand the most effective sample preparation method for studying the human serum proteome. A summary of the results will be presented.