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Molecular & Cellular Proteomics 5:1727-1744, 2006.
© 2006 by The American Society for Biochemistry and Molecular Biology, Inc.

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Challenges in Biomarker Discovery

Advances and Challenges in Liquid Chromatography-Mass Spectrometry-based Proteomics Profiling for Clinical Applications^*

Wei-Jun Qian, Jon M. Jacobs, Tao Liu, David G. Camp, II and Richard D. Smith

From the Biological Sciences Division and Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, Washington 99352

	ABSTRACT

TOP ABSTRACT CHALLENGES AND REQUIREMENTS FOR... ADVANCES IN LC-MS TECHNOLOGIES MULTIDIMENSIONAL FRACTIONATION... ANALYSIS THROUGHPUT CONFIDENCE OF PEPTIDE/PROTEIN... QUANTITATION STRATEGIES IMPLICATIONS OF HUMAN... TARGETED PROTEOMICS APPROACHES CONCLUSIONS AND PERSPECTIVES REFERENCES

 
Recent advances in proteomics technologies provide tremendous opportunities for biomarker-related clinical applications; however, the distinctive characteristics of human biofluids such as the high dynamic range in protein abundances and extreme complexity of the proteomes present tremendous challenges. In this review we summarize recent advances in LC-MS-based proteomics profiling and its applications in clinical proteomics as well as discuss the major challenges associated with implementing these technologies for more effective candidate biomarker discovery. Developments in immunoaffinity depletion and various fractionation approaches in combination with substantial improvements in LC-MS platforms have enabled the plasma proteome to be profiled with considerably greater dynamic range of coverage, allowing many proteins at low ng/ml levels to be confidently identified. Despite these significant advances and efforts, major challenges associated with the dynamic range of measurements and extent of proteome coverage, confidence of peptide/protein identifications, quantitation accuracy, analysis throughput, and the robustness of present instrumentation must be addressed before a proteomics profiling platform suitable for efficient clinical applications can be routinely implemented.

Proteomics-based candidate biomarker discovery efforts have recently gained significant attention due to the power of these technologies for analyzing complex protein mixtures and their potential for identifying novel markers indicative of disease. It is widely believed that many complex human diseases, including cancers, might be more effectively cured if specific disease biomarkers were available to enable detection and treatment at very early stages of disease (3). Despite noteworthy efforts, only a handful of cancer biomarkers have been approved by the United States Food and Drug Administration (FDA)¹ for clinical use, with the majority of these being protein biomarkers (4). Although existing markers play a significant role in screening, monitoring, and staging, effective biomarkers are not currently available for most cancers and are generally nonexistent for early detection (3). Therefore, there is a clear need for applying advanced technologies such as these based on proteomics in the quest for novel candidate clinical biomarkers.

Although widely speculated that advances in genomics and proteomics would alter the landscape of clinical biomarker discovery and validation, the declining trend of new FDA-approved biomarkers reported over the last decade (5) highlights the magnitude of the challenges associated with human clinical samples and validation of candidate biomarkers. Contributing to these challenges are the substantial complexity of the human proteome and the heterogeneity of the human population, both of which make the search for biomarkers from either biofluids or disease tissues a daunting task. As a result of the heterogeneous nature of humans and the complexity of diseases, e.g. cancers, a panel of biomarkers rather than a single marker may be required to achieve the high sensitivity and specificity required for clinical applications (3). Proteomics technologies offer significant potential for discovering such marker panels.

Many different technologies have been applied for biomarker discovery and other clinical applications, including two-dimensional (2D) gel-electrophoresis (6), LC-MS, and protein- and antibody-based microarrays (7–9). LC-MS- or tandem MS (MS/MS)-based proteomics technologies offer highly sensitive analytical capabilities and a relatively large dynamic range of detection and have increasingly become the method of choice for in depth profiling of complex protein mixtures (1). In addition, the relatively high throughput of LC-MS technologies is amenable to clinical applications that involve human biofluids and disease tissues. The application of LC-MS/MS for human biofluid protein profiling was initiated by the first global shotgun proteomics study of human plasma/serum published in 2002 by Adkins et al. (10). An explosion of LC-MS-based applications in human plasma/serum and various biofluids soon followed due to the tremendous interest in identifying disease-related proteins (11, 12). Various depletion/fractionation/enrichment techniques have been developed along the way and coupled to LC-MS to increase coverage of the biofluid proteomes (13).

Human blood serum/plasma remains the most commonly used clinical sample to date for proteomics applications because it may include specific biomarkers for virtually all human diseases due to its either direct or indirect interaction with the entire cell complement of the body, i.e. tissue-specific proteins may be released into the blood stream upon cell damage or cell death. Additionally serum/plasma can be readily obtained by clinical sampling. However, the magnitude of the previously mentioned challenges associated with human clinical samples coupled with the anticipation that potential biomarkers of interest could be present at extremely low concentrations in plasma has raised doubts as to whether disease biomarkers can be accurately detected or identified from plasma using a proteomics approach. As a result, analysis of various other biofluids/tissues has gained increasing attention. Due to their proximity to the source of disease or perturbation in the body, tissues (14) and various biofluids such as cerebrospinal fluid (15), bronchoalveolar lavage fluid (16), synovial fluid (17), nipple aspirate fluid (18), saliva (19), and urine (20) are believed to provide a more focused pool of potential biomarkers of interest. In addition, tumor interstitial fluids have also been reported as a novel source for proteomics biomarker and therapeutic target discovery (21), offering a promising alternative to direct tissue analysis. In the following review, we highlight LC-MS-based proteomics profiling for clinical applications by summarizing recent advances as well as the major challenges facing this technology for more effective candidate biomarker discovery.

	CHALLENGES AND REQUIREMENTS FOR DESIGNING A ROBUST LC-MS DISCOVERY PLATFORM

TOP ABSTRACT CHALLENGES AND REQUIREMENTS FOR... ADVANCES IN LC-MS TECHNOLOGIES MULTIDIMENSIONAL FRACTIONATION... ANALYSIS THROUGHPUT CONFIDENCE OF PEPTIDE/PROTEIN... QUANTITATION STRATEGIES IMPLICATIONS OF HUMAN... TARGETED PROTEOMICS APPROACHES CONCLUSIONS AND PERSPECTIVES REFERENCES

 
The distinctive nature of human biofluid proteomes, in particular the serum/plasma proteome, presents significant challenges for current analytical technologies aimed at quantitative protein profiling and biomarker discovery. First, the serum/plasma protein content is dominated by several very abundant proteins (i.e. the 22 most abundant proteins represent 99% of the total protein mass in plasma) yet at the same time presents an extraordinary dynamic range (>10 orders of magnitude) in protein concentrations that begins with serum albumin at 45 mg/ml and extends to cytokines (and potentially many disease-related proteins) at around 1–10 pg/ml or lower (5). Second, the serum/plasma proteome presents tremendous biological complexity as a result of tissue "leakage" proteins from the entire body, complex post-translational protein modifications such as glycosylation, and the existence of various forms (i.e. splice variants, proteolytic products, and the tremendous variability in the immunoglobulin class) for each expressed gene. Finally the substantial genetic and non-genetic biological variability of human clinical samples contributes significantly to the overall analytical challenge.

Despite significant recent advances, major challenges remain to prevent routine implementation of an LC-MS protein profiling platform suitable for efficient biomarker discovery (Table I). To effectively address these challenges, a protein profiling platform suitable for biomarker discovery and clinical applications must provide at the very minimum 1) overall high dynamic range of measurements and extensive coverage of the proteome for effective detection of low abundance proteins, 2) highly confident and specific protein identifications, 3) accurate quantitation of relative protein abundances across many clinical samples, and 4) high throughput capable of analyzing large numbers of clinical samples to provide sufficient statistical power needed to address biological variability. In addition, the platform, including both sample processing and LC-MS instrumentation, must be robust and include efficient informatics software capabilities for data mining and statistical analyses. Currently there is a broad consensus that no existing platform meets all of these requirements for effective biomarker discovery.

View larger version (19K): [in this window] [in a new window]   FIG. 1. A component diagram of an LC-MS protein profiling platform. FFE, free flow electrophoresis; 1D, one-dimensional; iTRAQ, isobaric tags for relative and absolute quantitation.

	ADVANCES IN LC-MS TECHNOLOGIES

TOP ABSTRACT CHALLENGES AND REQUIREMENTS FOR... ADVANCES IN LC-MS TECHNOLOGIES MULTIDIMENSIONAL FRACTIONATION... ANALYSIS THROUGHPUT CONFIDENCE OF PEPTIDE/PROTEIN... QUANTITATION STRATEGIES IMPLICATIONS OF HUMAN... TARGETED PROTEOMICS APPROACHES CONCLUSIONS AND PERSPECTIVES REFERENCES

To overcome the undersampling issue, our laboratory developed an accurate mass and time (AMT) tag approach that utilizes highly accurate mass measurements from a high resolution mass spectrometer (e.g. FTICR or TOF mass spectrometer) in conjunction with accurate elution time measurements from high resolution capillary LC separations to achieve high throughput proteome profiling without routine MS/MS measurements (29, 30). The concept of this AMT tag approach is based on the principle that the accurate mass and time measurements will allow reliable peptide identifications by correlating the mass and time of detected peaks to a pre-established peptide AMT tag reference library for a particular biological system (e.g. plasma). With this approach, LC-MS/MS proteome analyses coupled with extensive fractionation only need to be performed once to create an effective reference database of peptide markers defined by accurate masses and elution times, i.e. AMT tags. The AMT tag database then serves as a comprehensive "look-up table" for subsequent higher throughput LC-MS analyses, allowing many peptides in each spectrum to be identified without MS/MS. Fig. 2 exemplifies an LC chromatogram and 2D display of 2,800 peptides identified using the AMT tag strategy resulting from a single LC-FTICR analysis of a ProteomeLab^TM IgY-12 depleted human plasma sample.

View larger version (24K): [in this window] [in a new window]   FIG. 2. A typical LC-FTICR analysis of an IgY-12 depleted human plasma sample. A, the base peak chromatogram. B, a 2D display of 2,800 identified species at the mass and NET space. The analysis was performed using a Bruker 9.4-tesla FTICR instrument coupled with an LC system equipped with a 150-µm-inner diameter and 65-cm-long capillary column operated at 5,000 p.s.i.

As mentioned previously, the achievable dynamic range for the LC-MS platform depends significantly on the peak capacity of the on-line gradient reversed phase separations, the dynamic range of the MS system, and the efficiency and stability of the ESI interface. A single MS spectrum can provide a dynamic range of up to 10³ for a high resolution instrument (e.g. FTICR), and one would expect to achieve a dynamic range of at least 10⁵ by coupling this instrument to an on-line high resolution LC separation that provides a peak capacity of 1,000. However, the observed dynamic range of measurements can be significantly reduced for complex biological samples such as human plasma due to the charge competition of co-eluting high abundance species, leading to ion suppression of the relatively low abundance species. Ion suppression is a particular issue when analyzing human biofluid samples as these samples are dominated by a handful of highly abundant proteins. Significant ion suppression will occur when peptides originating from low abundance proteins of interest co-elute with peptides originating from high abundance proteins, leading to the inability to detect the co-eluting low abundance peptides.

Table II provides a summary of the relative proteome coverage and estimated dynamic ranges achieved by coupling high resolution reversed phase capillary LC separations with either MS/MS using an LTQ instrument or MS using a 9.4-tesla FTICR instrument. The enhanced coverage and dynamic ranges obtained by the removal of high abundance proteins and SCX fractionation are illustrated. All results shown in Table II are based on triplicate experiments that involved a pooled plasma sample from healthy subjects. The number of peptide identifications are reported with >95% confidence based on either a reversed database evaluation for MS/MS data (32) or a shifted database evaluation for the LC-FTICR data² with all proteins identified using a minimum of two different peptides. As shown, the single LC-MS/MS analysis only identifies 100 proteins with high confidence and provides a dynamic range of 10³. With the removal of either the top six (MARS) or top 12 (IgY-12) abundant proteins, the overall dynamic range is enhanced to 10⁵. LC-FTICR shows greater coverage for both peptide and protein identifications compared with LC-MS/MS, and the dynamic range is estimated to be similar to that observed for LC-MS/MS. (It should be noted that presently unassigned peptides probably include many more proteins.) When IgY-12 depletion and SCX fractionation are combined with LC-MS/MS, a dynamic range of 10⁶–10⁷ can be achieved, allowing identification of nearly 500 proteins in plasma with high confidence including many at the low ng/ml level, and 2D LC-FTICR analyses would be expected to increase this by approximately another order of magnitude. Note, however, that this dynamic range still falls 3 orders of magnitude short for detecting pg/ml protein concentrations. In addition, it should be noted that not all the proteins within the estimated dynamic range will be detected due to the differences in digestion efficiency and ion suppression effects for different proteins/peptides within the complex sample.

	MULTIDIMENSIONAL FRACTIONATION STRATEGIES COUPLED WITH LC-MS FOR IMPROVED PROTEOME COVERAGE

TOP ABSTRACT CHALLENGES AND REQUIREMENTS FOR... ADVANCES IN LC-MS TECHNOLOGIES MULTIDIMENSIONAL FRACTIONATION... ANALYSIS THROUGHPUT CONFIDENCE OF PEPTIDE/PROTEIN... QUANTITATION STRATEGIES IMPLICATIONS OF HUMAN... TARGETED PROTEOMICS APPROACHES CONCLUSIONS AND PERSPECTIVES REFERENCES

 
Given the tremendous dynamic range of protein abundances and the extraordinary complexity of human biofluid proteomes, many different fractionation techniques have been developed and applied in a multidimensional fashion to enhance dynamic range of detection and improve proteome coverage (13). Multicomponent immunoaffinity removal of highly abundant proteins in human plasma/serum (38, 39) has increasingly become the method of choice for prefractionating human plasma samples due to the high specificity, efficacy, and ease of coupling to other fractionation techniques. As shown in Table II, coupling the immunoaffinity depletion step to LC-MS provides an additional 1–2 orders of magnitude increase in dynamic range, allowing for detection of more low abundance proteins by effectively increasing the sample loading; similar improvements were reported in other studies (40, 41). Good reproducibility was demonstrated by performing immunoaffinity depletion with an automated LC system; however, some of the nontarget low abundance proteins have also been observed to bind to the columns but in a reproducible fashion (42). A possible approach to counter this effect is to analyze both the flow-through and bound fractions in more of a "partitioning" method instead of a pure "depletion" approach (39) with the accompanying trade-off of an increased number of required analyses. A further enhancement to the platform dynamic range will stem from the continuous improvement of antibody-based microbead technologies that will allow for removal of more highly to moderately abundant proteins.

Several different techniques for protein-level fractionation have been applied to human plasma/serum proteome profiling, including common gel-based techniques (43, 44), PF2D automated chromatofocusing/reversed phase LC (RPLC) (45) and other liquid chromatography-based separations (46), free-flow electrophoresis (41, 47), and IEF (46, 48–51). IEF is a common fractionation technique that has been applied to plasma profiling at both peptide and protein levels. Various forms of liquid phase IEF techniques have been developed, including off-gel electrophoresis (48), Rotofor (49) or MiniRotofor (46), microscale solution IEF (ZOOM) (50), and a preparative multichannel electrolyte system (51). A common feature of these systems is the multiple tandem electrode chambers used to partition complex protein samples. IPG IEF followed by in-gel digestion has also been used for plasma protein fractionation prior to LC-MS/MS (52). A number of recent large scale proteome profiling studies have combined different protein- and peptide-level fractionation techniques (e.g. PF2D (45), SCX/RPLC (54), free flow electrophoresis-IEF/RPLC (47), ZOOM/SDS-PAGE (50), and Rotofor/RPLC/SDS-PAGE (49) protein fractionation) with peptide-level LC-MS/MS analyses to achieve more comprehensive coverage of the plasma proteome.

An alternative to plasma protein fractionation is to specifically enrich functional "subproteomes" such as the glycoproteome or the cysteinyl subproteome by using chemical tagging or capture agents; this significantly reduces overall sample complexity and enhances detection of low abundance proteins. For example, we have recently demonstrated a simple procedure for effectively enriching cysteinyl peptides from complex proteomes (including human biofluids (55)) that provides significantly improved proteome coverage when used as a peptide-level fractionation technique (27). Additionally hydrazine chemistry can be applied to specifically enrich N-linked glycopeptides (56, 57), and multilectin affinity chromatography can be used to isolate and characterize glycoproteins from human plasma and serum samples (58). Our laboratory has recently developed a strategy that combines immunoaffinity depletion and subsequent chemical fractionation based on cysteinyl peptide and N-glycoprotein captures with 2D LC-MS/MS for in depth plasma profiling (Fig. 3 ) (59). Application of this "divide-and-conquer" strategy to trauma patient plasma samples resulted in confident identification of 1,500 different proteins (with a minimum of two peptides per protein; 99.5% confidence level based on reversed database evaluation) and illustrated an overall dynamic range of detection of >10⁷ (low ng/ml concentrations for six identified low abundance proteins were verified by ELISA).

View larger version (20K): [in this window] [in a new window]   FIG. 3. Schematic representation of a chemical fractionation strategy applied to the plasma proteome characterization. High abundance proteins were first removed using immunoaffinity subtraction. The resulting less abundant proteins were split and subjected to solid phase cysteinyl peptide and N-glycoprotein captures independently. Non-cysteinyl peptides and non-glycopeptides generated at the same time were also collected. All four different peptide populations were then fractionated by SCX, and each fraction was analyzed by capillary LC-MS/MS. PNGase F, peptide-N-glycosidase F (59).

	ANALYSIS THROUGHPUT

TOP ABSTRACT CHALLENGES AND REQUIREMENTS FOR... ADVANCES IN LC-MS TECHNOLOGIES MULTIDIMENSIONAL FRACTIONATION... ANALYSIS THROUGHPUT CONFIDENCE OF PEPTIDE/PROTEIN... QUANTITATION STRATEGIES IMPLICATIONS OF HUMAN... TARGETED PROTEOMICS APPROACHES CONCLUSIONS AND PERSPECTIVES REFERENCES

Several recent technology developments hold potential for greatly enhancing the overall analysis throughput of clinical samples. The first is the development of very fast LC separations for proteomics analyses. Current automated LC-MS proteomics platforms typically involve LC separations with gradients of 100 min or longer, which limits throughput to 10 sample analyses per day per MS instrument. Several reports have explored the use of smaller particle-packed columns or monolithic columns for fast LC separations (10 min or less) as well as multiplex column systems to significantly improve the throughput (60, 61). However, it is unclear whether sufficient separation power can be achieved with these fast liquid phase separations because the increase in the solvent gradient speed can degrade the separation peak capacity (60), which in turn reduces the overall dynamic range of detection. Other strategies for achieving robust fast separations include liquid phase chromatographic and electrophoretic separations on a microfluidic chip platform (62–64). Such chip-based separation devices also have the advantage of providing better robustness, reliability, and ease of operation.

Very fast (millisecond scale) gas phase separations based on ion mobility spectrometry (IMS; a separation method that is somewhat analogous to electrophoresis in the gas phase) are another powerful alternative to liquid phase separations for significant improvement in throughput. At its simplest, an IMS stage consists of a drift tube filled with a non-reactive gas (commonly helium or N₂) and a uniform electric field established along the axis of separation. Mixtures of peptides, proteins, or small molecules are separated by their gas phase cross-sections (size) in addition to charge, and knowledge of their mobility provides another separation dimension to aid in identification.

The power of IMS has been advanced by several recent technical developments. IMS coupled with a TOF MS platform and combinatorial libraries (65) has been recently demonstrated for analysis of proteolytic digests (66). Because an IMS separation typically requires 1–100 ms and has a resolving power of 50–200, a single species IMS peak exits the drift tube over a 0.1–1-ms period. Generation of a typical TOF MS spectrum requires 30–100 µs, which allows multiple mass spectra to be obtained during the "elution" of an IMS peak. More recently, LC has been coupled to IMS-TOF MS via an ESI interface, providing 2D separations prior to MS analysis (67). Despite enormous potential for high throughput analyses of complex samples, the application of IMS-TOF MS has been limited by low sensitivity due to ion losses at the IMS-MS interface; however, the recent implementation of electrodynamic ion funnels at both the ESI-IMS and IMS-TOF MS interfaces has significantly improved the sensitivity of the overall LC-ESI-IMS-TOF MS platform (Fig. 4 ) (68) such that the sensitivity is now comparable to that of a commercial ESI-MS instrument. Although still in the development stage, the very fast separation speed and potential high dynamic range of measurements offered by the 2D liquid phase-gas phase separations make LC-ESI-IMS-TOF MS an attractive and practical platform for high throughput clinical applications.

View larger version (16K): [in this window] [in a new window]   FIG. 4. Schematic diagram of a prototype ESI-IMS-Q-TOF instrumentation platform that uses electrodynamic ion funnel interfaces at both ends of the IMS drift tube and, as a result, provides very high sensitivity from high speed analyses. Reproduced with permission from Ref. 68, copyright 2005 Am. Chem. Soc.

	CONFIDENCE OF PEPTIDE/PROTEIN IDENTIFICATIONS

TOP ABSTRACT CHALLENGES AND REQUIREMENTS FOR... ADVANCES IN LC-MS TECHNOLOGIES MULTIDIMENSIONAL FRACTIONATION... ANALYSIS THROUGHPUT CONFIDENCE OF PEPTIDE/PROTEIN... QUANTITATION STRATEGIES IMPLICATIONS OF HUMAN... TARGETED PROTEOMICS APPROACHES CONCLUSIONS AND PERSPECTIVES REFERENCES

To address the issue of false peptide identifications, we recently performed a probability-based evaluation of peptide identifications derived from LC-MS/MS and SEQUEST analysis in which selected human proteomes, including human plasma, were searched against a sequence-reversed human protein database (32) similar to a previous report applying the reversed database strategy to the yeast proteome (72). The reversed protein database was created by reversing the order of amino acid sequences for each protein (the carboxyl terminus becomes the amino terminus and vice versa) in the original human protein database. This approach assumes that the numbers of false positives that arise from "random" hits should be the same for both the normal database and the reversed database because the reversed database is identical in number of protein entries, protein size, and distribution of amino acids to the normal database. Fig. 5 shows a histogram of Xcorr distribution for unique peptides (charge state 2+; fully tryptic) from a human plasma sample identified by searching the normal (solid line) and reversed (dashed line) databases. The Xcorr distribution allows an estimated confidence level for any given Xcorr bin as well as the overall false positive rate for a given Xcorr cutoff to be calculated by dividing the area beneath the dashed line (reversed database hits) by the area beneath the solid line (normal database hits) for a given Xcorr range. This study also revealed the high false positive rates for plasma/serum peptide/protein identifications in several previously published studies (10, 69, 70, 73, 74). For example, 30% false positives were observed when the often cited Washburn et al. (75) filtering criteria were applied to human plasma. Thus, filtering criteria that provided overall >95% confidence at the unique peptide level for both human cell lines and human plasma were proposed. When identical filtering criteria were used, the observed false positive rates of peptide identifications for human plasma were significantly higher than those for the human cell lines, suggesting that the false positive rates are significantly dependent upon sample characteristics, particularly the number of proteins found within the detectable dynamic range for different samples. Additionally Xie and Griffin (76) reported the increased potential for false positive identifications for the 2D linear ion trap (LTQ) when compared with a traditional three-dimensional ion trap (LCQ) instrument, and more stringent filtering criteria are required for LTQ compared with LCQ to minimize false positive identifications. These results suggest that peptide/protein identification confidence levels not only depend on sample characteristics but also on components of the LC-MS platform.

View larger version (16K): [in this window] [in a new window]   FIG. 5. Relative frequency of different peptides identified from the normal human protein database (solid line) and the reversed human protein database (dashed line) at different Xcorr values. Data shown are for the 2+ charge state fully tryptic peptides identified from human plasma and filtered with Cn 0.1. Reproduced with permission from Ref. 32, copyright 2005 Am. Chem. Soc.

PeptideProphet provides another independent statistical model for evaluating potential false positive peptide identifications. The model utilizes the expectation maximum algorithm to derive a mixture of correct and incorrect peptide assignments from the data (79). This approach has been directly compared with the reversed database approach for analyzing the same dataset derived from human plasma (59). Following filtering with reversed database criteria, 6,279 unique peptides were identified from this dataset with >95% confidence, whereas 6,341 unique peptides were identified by PeptideProphet using a minimum computed probability of 0.95. Approximately 95% of peptides were common between the two datasets, suggesting comparable results from these two statistical approaches. The use of ProteinProphet, another statistical model that computes the probability of the presence of proteins, addresses the issue of whether peptides are present in more than one entry in the protein database (protein redundancy problem) (80). The list of identified peptides from both the PeptideProphet and the reversed database filtering approaches can serve as input for ProteinProphet to generate a list of non-redundant protein identifications. Several other statistical methods have been recently described for evaluating peptide assignments from MS/MS spectra (81–83). Ideally universal acceptance of a statistical model that optimizes both sensitivity and specificity for confident peptide identifications from MS/MS spectra will allow cross-comparison of protein profiling results from different laboratories, which currently remains as an unresolved challenge.

Similar challenges exist for evaluating false positive identifications from MS-only approaches that utilize accurate mass measurements for peptide/protein identifications. The utility of accurate mass measurements initially was demonstrated in the "peptide mass fingerprinting" approach for protein identification in which a set of peptide fragments unique to each protein are created by digestion, and the mass of these peptide fragments is used as a "fingerprint" to identify the original protein (84–86). Thus far, this approach has been limited to simple protein mixtures or single proteins. The more recently reported AMT tag approach utilizes accurate LC retention time measurements in addition to accurate mass measurements to identify peptides and has been successfully applied to global proteome profiling, including the human plasma proteome (31, 87). With the AMT tag approach, peptides are identified by matching LC-MS observed mass and normalized elution time (NET) features to AMT tags in the pre-established reference database (look-up table of peptides) with a given mass error and NET error tolerances (typically 1–5 ppm for mass and 1–3% for NET). The potential false positive identifications resulting from random matching of features to the reference database are indicated on histograms of mass error (the difference between observed mass and calculated mass for the matched peptide in the database) exemplified in Fig. 6 A for a human plasma dataset analyzed by LC-FTICR. Note that the use of the NET constraint significantly reduces the level of random matches as indicated by the background level for each histogram. Similar to the reversed database approach for MS/MS, we have recently applied a shifted database approach for evaluating the false positive rate in the AMT tag process.² As shown in Fig. 6B, an 3% false positive rate for this human plasma dataset was estimated as the ratio of the area beneath the curve that represents matches to the shifted database (black squares) and the area beneath the curve that represents matches to the normal database within a ±2 ppm window (gray circles). In addition to being used for direct identification in the MS-only approach, the accurate mass information also has been utilized for improving the confidence of peptide identifications by MS/MS through application of the new generation of LTQ-FT and LTQ-Orbitrap mass spectrometers (88, 89).

View larger version (19K): [in this window] [in a new window]   FIG. 6. A, mass error histograms of features detected from a single LC-FTICR dataset of a human plasma sample that matched to a human plasma AMT tag database using different levels of NET constraints. The LC separation time is normalized to a 0–1 scale in NET. B, mass error histograms for features from the same dataset matching to a normal AMT tag database (gray circles) and to a shifted AMT tag database (black squares). Note, the black squares represent random matches to the 11 Da shifted AMT tag database.

	QUANTITATION STRATEGIES

TOP ABSTRACT CHALLENGES AND REQUIREMENTS FOR... ADVANCES IN LC-MS TECHNOLOGIES MULTIDIMENSIONAL FRACTIONATION... ANALYSIS THROUGHPUT CONFIDENCE OF PEPTIDE/PROTEIN... QUANTITATION STRATEGIES IMPLICATIONS OF HUMAN... TARGETED PROTEOMICS APPROACHES CONCLUSIONS AND PERSPECTIVES REFERENCES

More recently, we have demonstrated ¹⁶O/¹⁸O labeling combined with the AMT tag strategy as an effective global quantitative approach for quantifying relative protein abundance differences in human plasma (31). By incubating tryptic peptides in ¹⁸O water (55, 94) in the presence of trypsin, the ¹⁸O atoms are incorporated into the carboxyl terminus of tryptically cleaved peptides via a postdigestion trypsin-catalyzed oxygen exchange reaction. The ¹⁶O/¹⁸O-labeled peptide pairs provide a 4-Da mass difference (Fig. 7 A), which allows a high resolution mass spectrometer such as FTICR or TOF to effectively resolve the ¹⁶O- and ¹⁸O-labeled peptide pairs and accurately measure the relative abundances. The advantage is that all types of samples (e.g. tissues, cells, and biological fluids) can be effectively labeled using this simple and specific enzyme-catalyzed reaction. Fig. 7A shows a partial 2D display of detected peptide pairs in mass versus time dimensions. The ¹⁸O/¹⁶O-labeled peptides are readily visualized as co-eluting pairs (4 Da apart), and the abundance ratio can be precisely calculated for each ¹⁸O/¹⁶O pair. In this initial comparative analysis demonstration of two human plasma samples obtained from a healthy individual prior to (control) and following LPS administration, relative abundance differences between the two plasma samples were quantified for a total of 429 plasma proteins. Fig. 7B shows the normalized -fold changes in 429 quantified proteins and demonstrates the significant changes in abundance for a set of proteins following LPS administration. The combined ¹⁶O/¹⁸O labeling-AMT tag strategy can also be easily coupled with subsequent peptide-level fractionation approaches such as cysteinyl peptide enrichment (55) and SCX fractionation.

View larger version (11K): [in this window] [in a new window]   FIG. 7. A, a partial 2D display of the detected 18O/16O-labeled peptide pairs from an LC-FTICR analysis. The elution time is shown as a normalized scale between 0 and 1. Observed peaks (represented by spots) correspond to various eluting peptides. The heavy and light isotope-labeled pairs are easily visualized with a 4-Da mass difference. B, normalized -fold changes for the 429 quantified proteins following LPS administration. The abundance ratio for each protein shown was normalized to zero (R – 1) (53). For ratios smaller than 1, normalized inverted ratios were calculated as 1 – (1/R). The error bar for each protein indicates the S.D. for the abundance ratios from multiple peptides. Proteins without error bars were identified with single peptides.

Alternatively "label-free" direct quantitation approaches hold interest because of greater flexibility for comparative analyses and simpler sample processing procedures compared with labeling approaches. The isotope labeling and label-free approaches are complementary, and each approach has different sources of variations. Several initial studies suggest that the use of normalized LC-MS peak intensities for detected peptides can be used to compare relative abundances between similar complex samples (101–103). It has been demonstrated that abundance ratios of separate model proteins may be predicted to within 20% in complex proteome digests by using measured peptide ion intensities obtained in LC-MS analyses (101). Among the main challenges for label-free quantitation are the multiple issues that affect the usefulness of peptide peak intensities for relative quantitation, such as differences in electrospray ionization efficiencies among different peptides and different samples (37), differences in the amount of sample injected in each analysis, and sample preparation reproducibility. These issues are often peptide-dependent, leading to observed disparity among relative abundances of different peptides originating from the same protein. The significant bias and ion suppression effects caused by charge competition (ionization bias) during ESI (104) are often considered a major limitation for accurate label-free quantitation. Recent studies have demonstrated substantial advantages for ESI-MS analyses at nanoflow regimes (<100 nl/min) afforded by narrower inner diameter capillary columns for separations (36, 37). It is well demonstrated that smaller inner diameter columns with lower flow rates provide significantly higher sensitivity than larger inner diameter columns with higher flow rates (34) because of the significant improvements in both ionization and MS sampling efficiencies. Reversed phase packed nanoscale LC and monolithic nanoscale LC separations have been developed and coupled to ESI for improved ionization and quantitation (34, 105). As ionization efficiencies are increased for nanoelectrospray, detection biases are decreased because undesired matrix effects and/or ion suppression effects are either reduced or eliminated (104–106), providing the basis for improved quantitation. With further improvements to the robustness of these nano-LC-ESI-MS systems, label-free quantitation may be widely applied in clinical applications.

Another challenge for quantitative clinical proteomics applications is the variability introduced during multiple steps of sample processing. With continued development of cleanup products for more consistent performance and automated sample processing, such reproducibility issues may be minimized, leading to further improvements in quantitation when applying either the stable isotope labeling or label-free approaches.

	IMPLICATIONS OF HUMAN HETEROGENEITY IN CLINICAL PROTEOMICS STUDIES

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The ability to identify disease-specific differences by using a proteomics approach relies on multiple factors integral to the overall analysis pipeline. For example, when performing peptide-level measurements, achieving high peptide identification quality is a prerequisite for assuring confidence in all other downstream parameters (i.e. confidence in both protein identification and quantitation), whereas the ability to quantify differences between any two samples largely depends on the reproducibility of the overall platform. Due to inherent variations that stem from sample preparation and instrument analysis, technical replicates are often performed to evaluate and minimize technical variability arising from the overall analysis pipeline. Technical variability will be minimized as technologies continue to mature, and platforms will likely become more robust and reproducible; however, biological variability within the same comparative groups remains as a challenge for identifying real differences between different conditions. Although ideally one would like to either control or minimize such biological variability by utilizing more controlled model systems such as cell cultures, an in vitro model system, or even inbred mouse strains, this is not always possible. Most clinical studies are based on "real world" human clinical samples where inherent human individual heterogeneity makes discovery efforts more difficult. The human heterogeneity challenge in proteomics studies stems from the high probability that two equally "healthy" individuals will have overall significantly different individual protein abundance levels when sampled at any given time. This heterogeneity can be due to individual genetic variability (i.e. gender, race, etc.) and/or to contributing environmental factors such as diet, overall health, detrimental environmental exposures, etc. The complexity of human diseases presents another degree of challenge. For example, in human cancer, each tumor type typically consists of a number of subtypes that differ with regard to their spectrum of genetic alterations (107). Therefore, a potential candidate biomarker of disease may be elevated only in a certain percentage of the pool of disease patients.

The implications of human heterogeneity in the context of LC-MS-based proteomics experiments centers mostly on the measured quantitative values for peptide/protein identifications. Fig. 8 shows an initial evaluation of the technical variation and biological variations of human and mouse plasma samples based on the Pearson correlation of the identified peptide intensities between any two individual samples. The technical replicate results (Fig. 8A; nine individually processed samples from one pooled reference plasma) show overall good correlation (0.94 ± 0.02), which suggests relatively good reproducibility of the overall analytical platform. The increased variation among human subjects (Fig. 8B) appears obvious on the basis of significantly reduced average correlation coefficients (0.85 ± 0.06) compared with the technical replicate results; whereas mouse plasma samples (Fig. 8C) show only slightly reduced correlation (0.92 ± 0.05), which suggests relatively small biological variation in these inbred mouse models. Such large variations observed among different healthy control subjects present a challenge for identifying disease-specific differences. To address these challenges and increase the confidence of discovery results, it is essential for the discovery platform to be able to analyze a relatively large number of clinical samples in a high throughput manner to obtain sufficient statistical power.

View larger version (45K): [in this window] [in a new window]   FIG. 8. Pearson correlation plot comparing peptide intensities of LC-FTICR analyses of plasma samples. A, nine technical replicates for a pooled reference human plasma sample from multiple healthy subjects. B, nine human plasma samples from individual healthy subjects with ages range from 18 to 26. C, nine mouse plasma samples isolated from individual C57BL6 mice. Each sample including the technical replicate was separately processed by ProteomeLab IgY-12 (for human) or IgY-R7 (for mouse) depletion, and the flow-through portions were digested with trypsin prior to LC-MS analyses.

Designing experiments to minimize biological variability is imperative for clinical studies. One example is to analyze a serial sample set, i.e. plasma or biopsy tissue samples, from the same individual over a time course or disease progression; this in theory will alleviate a majority of heterogeneity effects, but such samples are traditionally more difficult to obtain in addition to the fact that most patients do not have a "control" blood or tissue sample in storage for comparison against a possible disease diagnosis. For most studies that use cross-sectional approaches, it is desirable to match the patients and controls in terms of age, sex, race, weight, and even diet if possible. A recent study reported the potential utility of pooling for reducing the effects of biological variation in microarray studies while retaining the accuracy of identifying differentially expressed genes when biological replicates are retained in the study design and providing the additional benefit of a great reduction in the total number of samples to be analyzed (110). Such a strategy might be explored and extended to clinical proteomics studies.

A further implication in heterogeneity is the presence of protein isoforms, splice variants, specific amino acid mutations, proteolytic products, and other post-translational modifications that are likely present in individual samples but are most often not explicitly included as sequences in the searchable protein database. This exclusion makes it challenging for traditional LC-MS/MS-based bottom-up approaches to identify such modified proteins and is possibly one of the main reasons that a large percentage of MS/MS spectra in clinical analyses remain unidentified. The identification of amino acid-specific post-translational modifications (e.g. phosphorylation, glycosylation, glycation, nitration, oxidation, and deamination) challenges MS/MS-based approaches due to the vast variety of possible modifications and the potential high false positive rates that originate from database searching. Because it is recognized that many protein biomarkers may be specific protein isoforms or modified proteins, further technical developments for more effective identification and quantitation of protein isoforms and modifications would be greatly desirable.

As an alternative to identifying protein isoforms and modifications, intact protein-level separations can be used to separate different protein isoforms on the basis of their different masses or other properties. The ability to use 2D gel electrophoresis for resolving different isoforms and monitoring their abundance changes has been well documented (111). The recently developed multidimensional intact protein analysis system (IPAS) separates intact proteins on the basis of charge, hydrophobicity, and molecular mass; quantitation is achieved by protein tagging with fluorophores (43). The potential for revealing different protein isoforms and specific protein cleavage products in human plasma/serum also has been demonstrated (49). The advantages offered by intact protein analysis complements the bottom-up proteomics approaches, and better integration of these two approaches may lead to more effective biomarker discovery.

	TARGETED PROTEOMICS APPROACHES

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The majority of proteomics applications in the search for candidate biomarkers to date have been focused on global proteome characterization focused on identifying multiple protein differences (candidate biomarkers) that correlate with specific human diseases; however, as discussed previously, there are many challenges associated with applying such a strategy to the discovery of low abundance candidate marker proteins. An alternative strategy for biomarker discovery that complements global profiling is the targeted proteomics approach that involves quantitative MS to measure a hypothesis-generated list of candidates (112). The targeted proteomics strategy often provides greater sensitivity and allows for detection of low abundance candidate proteins. Anderson and Hunter (113) recently demonstrated the use of peptide multiple reaction monitoring (MRM) for quantitative assaying of major plasma proteins. Such MRM assays provide great specificity for peptide/protein identifications and relatively good precision for quantitation. Additionally MRM can provide a rapid and specific platform for biomarker validation, particularly when coupled with specific enrichment techniques such as the recently published SISCAPA (Stable Isotope Standards and Capture by Anti-Peptide Antibodies) method for enriching target peptides using anti-peptide antibodies (114). Activity-based protein profiling is another strategy that uses chemical probes for tagging, enriching, and isolating a specific subset of physiologically important proteins on the basis of enzymatic activity (115, 116). Coupling such strategies with LC-MS holds potential for eliminating many issues related to the dynamic range of protein abundance.

A continuing issue for current LC-MS-based profiling approaches is that many of the detected species or features from LC-MS and LC-MS/MS analyses remain unidentified. Based on our experience, 80% of MS/MS spectra on average are not confidently identified via database searching, and more than 50% of LC-FTICR-detected features remain unidentified by the AMT tag approach. Present informatics tools and statistical algorithms have been able to utilize intensity information of these unidentified features to identify "interesting" features as potential biomarkers for specific diseases; effectively targeting these interesting features using data-directed or targeted MS/MS approaches is of current interest. One of the informatics challenges associated with identifying these features concerns different post-translational modifications. Current commercial mass spectrometers such as the LTQ offer a targeted MS/MS capability based on the selection of a list of m/z values. Developing an advanced targeted MS/MS approach (117) that incorporates "smart selection" of the targets and different, but complementary fragmentation techniques will be an integral component for an effective LC-MS profiling platform suitable for clinical applications.

	CONCLUSIONS AND PERSPECTIVES

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The amount of effort placed into the development and application of effective proteomics profiling of serum/plasma and other clinical samples has increased tremendously over the last several years. With the emergence of more effective LC-MS technologies and the variety of fractionation approaches, the number of proteins detectable in human plasma by global profiling has been greatly expanded (e.g. 889 proteins with >95% confidence reported in the recent HUPO study and 1,494 proteins with >99% confidence, including confident identification of many low ng/ml level plasma proteins, in our recent study (59)). Although this level of detection still falls short of the 10 orders of magnitude in dynamic range that encompasses plasma protein abundances, it still offers significant potential for the discovery of novel candidate biomarkers from clinical plasma/serum samples.

Currently there is no single platform that represents the "best" technology for such discovery applications, and integration of multiple technologies is often required for detection and quantitation of low abundance proteins. The need for improved reproducibility, throughput, dynamic range, and quantitation will continue to drive technology development and improvement efforts. Importantly several new technological developments such as fast LC separations, gas phase IMS separations, and high efficiency nano-ESI interfaces presently appear promising for future discovery platforms and applications. With improvements in quantitation accuracy, throughput, and robustness, the LC-MS protein profiling platform may eventually become a powerful tool for clinical diagnostic testing that provides simultaneous measurements of a large number of clinically relevant analytes.

An important component of any integrated profiling platform not previously discussed is the informatics and statistical analysis. The development of more effective software packages will be essential for processing the large number of LC-MS datasets, which may include peak (or feature) detection, run-to-run feature alignment, intensity normalization, feature matching to the database, and statistical analysis to generate a list of high confidence potential candidates.

Finally due to the complexity of large scale clinical proteomics studies, collaborative efforts from multiple laboratories with different platforms may be required for benchmarking and better cross-validation of the discovery results and eliminating potential biases introduced into any given platform. This implies that a common set of standards is needed so that platform performance in different laboratories may be readily compared and large scale proteomics datasets can be effectively exchanged and shared.

	ACKNOWLEDGMENTS

 
The contributions of Marina Gritsenko, Hongliang Jiang, Matt Monroe, Ron Moore, Tom Metz, Angela Norbeck, Sam Purvine, and Yufeng Shen to the work reviewed here are gratefully acknowledged.

	FOOTNOTES

 
Received, May 2, 2006, and in revised form, July 25, 2006.

Published, MCP Papers in Press, August 3, 2006, DOI 10.1074/mcp.M600162-MCP200

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ The abbreviations used are: FDA, Food and Drug Administration; SCX, strong cation exchange chromatography; NET, normalized elution time; AMT, accurate mass and time; IMS, ion mobility spectrometry; 2D, two-dimensional; RPLC, reversed phase LC; MARS, multiple affinity removal system; HUPO, Human Proteome Organization; LPS, lipopolysaccharide; MRM, multiple reaction monitoring.

² V. A. Petyuk, W. J. Qian, M. H. Chin, H. Wang, E. A. Livesay, M. E. Monroe, J. N. Adkins, N. Jaitly, D. J. Anderson, D. G. Camp, D. J. Smith, and R. D. Smith, manuscript submitted.

^* Portions of the reviewed research were supported by the United States Department of Energy (DOE) Office of Biological and Environmental Research; the National Institutes of Health through the National Center for Research Resources Grant RR018522, NIGMS Large Scale Collaborative Research Grant U54 GM-62119-02, NIDDK Grant R21 DK070146, and NIDA Grant 1P30DA01562501; the Entertainment Industry Foundation (EIF) and the EIF Women’s Cancer Research Fund; and the Laboratory Directed Research Development program at Pacific Northwest National Laboratory. Our laboratories are located in the Environmental Molecular Sciences Laboratory, a national scientific user facility sponsored by the DOE and located at Pacific Northwest National Laboratory, which is operated by Battelle Memorial Institute for the DOE under Contract DE-AC05-76RL0 1830.

To whom correspondence should be addressed: Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, P.O. Box 999, MSIN: K8-98, Richland, WA 99352. E-mail: rds{at}pnl.gov

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