Less label, more free: approaches in label-free quantitative mass spectrometry

In this review we examine techniques, software, and statistical analyses used in label-free quantitative proteomics studies for area under the curve and spectral counting approaches. Recent advances in the field are discussed in an order that reflects a logical workflow design. Examples of studies that follow this design are presented to highlight the requirement for statistical assessment and further experiments to validate results from label-free quantitation. Limitations of label-free approaches are considered, label-free approaches are compared with labelling techniques, and forward-looking applications for label-free quantitative data are presented. We conclude that label-free quantitative proteomics is a reliable, versatile, and cost-effective alternative to labelled quantitation.     

Trends in mass spectrometry instrumentation for proteomics

Mass spectrometry has become a primary tool for proteomics because of its capabilities for rapid and sensitive protein identification and quantitation. It is now possible to identify thousands of proteins from microgram sample quantities in a single day and to quantify relative protein abundances. However, the need for increased capabilities for proteome measurements is immense and is now driving both new strategies and instrument advances. These developments include those based on integration with multi-dimensional liquid separations and high accuracy mass measurements and promise more than order of magnitude improvements in sensitivity, dynamic range and throughput for proteomic analyses in the near future.     

Quantitative proteomics using mass spectrometry

The use of stable isotopes as internal standards in mass spectrometry has opened a new era for quantitative proteomics. Depending on the point at which the label is introduced, most procedures can be classified as in vivo labeling, in vitro pre-digestion labeling or in vitro post-digestion labeling. In vivo labeling has been used for cells that can be grown in culture and has the advantage of being more accurate. The pre-digestion and post-digestion labeling procedures are suitable for all types of sample including human body fluids and biopsies. Several new mass spectrometric strategies mark significant achievements in determining relative protein concentrations and in quantifying post-translational modifications. However, further technology developments are needed for understanding the complexity of a dynamic system like the proteome.     

Utility of gel-free,label-free shotgun proteomics approaches to investigate microorganisms

This review will examine the current situation with label-free, quantitative, shotgun-oriented proteomics technology and discuss the advantages and limitations associated with its capability in capturing and quantifying large portions of proteomes of microorganisms. Such an approach allows (1) comparisons between physiological or genetic states of organisms at the protein level, (2) ‘painting’ of proteomic data onto genome data-based metabolic maps, (3) enhancement of the utility of genomic data and finally (4) surveying of non-genome sequenced microorganisms by taking advantage of available inferred protein data in order to gain new insights into strain-dependent metabolic or physiological capacities. The technology essentially is a powerful addition to systems biology with a capacity to be used to ask hypothesis-driven ‘top-down’ questions or for more empirical ‘bottom-up’ exploration.     

Clinical proteomics and mass spectrometry profiling for cancer detection

A key challenge in the clinical proteomics of cancer is the identification of biomarkers that would enable early detection, diagnosis and monitoring of disease progression to improve long-term survival of patients. Recent advances in proteomic instrumentation and computational methodologies offer a unique chance to rapidly identify these new candidate markers or pattern of markers. The combination of retentate affinity chromatography and mass spectrometry is one of the most interesting new approaches for cancer diagnostics using proteomic profiling. This review presents two technologies in this field, surface-enhanced laser desorption/ionization time-of-flight and Clinprot?, and aims to summarize the results of studies obtained with the first of them for the early diagnosis of human cancer. Despite promising results, the use of the proteomic profiling as a diagnostic tool brought some controversies and technical problems, and still requires some efforts to be standardized and validated.     

Clinical proteomics and mass spectrometry profiling for cancer detection

A key challenge in the clinical proteomics of cancer is the identification of biomarkers that would enable early detection, diagnosis and monitoring of disease progression to improve long-term survival of patients. Recent advances in proteomic instrumentation and computational methodologies offer a unique chance to rapidly identify these new candidate markers or pattern of markers. The combination of retentate affinity chromatography and mass spectrometry is one of the most interesting new approaches for cancer diagnostics using proteomic profiling. This review presents two technologies in this field, surface-enhanced laser desorption/ionization time-of-flight and Clinprot, and aims to summarize the results of studies obtained with the first of them for the early diagnosis of human cancer. Despite promising results, the use of the proteomic profiling as a diagnostic tool brought some controversies and technical problems, and still requires some efforts to be standardized and validated.     

A noise model for mass spectrometry based proteomics

Motivation: Mass spectrometry data are subjected to considerablenoise. Good noise models are required for proper detection andquantification of peptides. We have characterized noise in bothquadrupole time-of-flight (Q-TOF) and ion trap data, and haveconstructed models for the noise. Results: We find that the noise in Q-TOF data from Applied BiosystemsQSTAR fits well to a combination of multinomial and Poissonmodel with detector dead-time correction. In comparison, iontrap noise from Agilent MSD-Trap-SL is larger than the Q-TOFnoise and is proportional to Poisson noise. We then demonstratethat the noise model can be used to improve deisotoping forpeptide detection, by estimating appropriate cutoffs of thegoodness of fit parameter at prescribed error rates. The noisemodels also have implications in noise reduction, retentiontime alignment and significance testing for biomarker discovery. Contact: pdu{at}us.ibm.com Supplementary information: Supplementary data are availableat Bioinfomatics Online. Associate Editor: Olga Troyanskaya     

Charge state estimation for tandem mass spectrometry proteomics

High-throughput protein analysis by tandem mass spectrometry produces anywhere from thousands to millions of spectra that are being used for peptide and protein identifications. Though each spectrum corresponds only to one charged peptide (ion) state, repetitive database searches of multiple charge states are typically conducted since the resolution of many common mass spectrometers is not sufficient to determine the charge state. The resulting database searches are both error-prone and time-consuming. We describe a straightforward, accurate approach on charge state estimation (CHASTE). CHASTE relies on fragment ion peak distributions, and by using reliable logistic regression models, combines different measurements to improve its accuracy. CHASTE's performance has been validated on data sets, comprised of known peptide dissociation spectra, obtained by replicate analyses of our earlier developed protein standard mixture using ion trap mass spectrometers at different laboratories. CHASTE was able to reduce number of needed database searches by at least 60% and the number of redundant searches by at least 90% virtually without any informational loss. This greatly alleviates one of the major bottlenecks in high throughput peptide and protein identifications. Thresholds and parameter estimates can be tailored to specific analysis situations, pipelines, and instrumentations. CHASTE was implemented in Java GUI-based and command-line-based interfaces.     

Decision tree-driven tandem mass spectrometry for shotgun proteomics

Mass spectrometry has become a key technology for modern large-scale protein sequencing. Tandem mass spectrometry, the process of peptide ion dissociation followed by mass-to-charge ratio (m/z) analysis, is the critical component for peptide identification. Recent advances in mass spectrometry now permit two discrete, and complementary, types of peptide ion fragmentation: collision-activated dissociation (CAD) and electron transfer dissociation (ETD) on a single instrument. To exploit this complementarity and increase sequencing success rates, we designed and embedded a data-dependent decision tree algorithm (DT) to make unsupervised, real-time decisions of which fragmentation method to use based on precursor charge and m/z. Applying the DT to large-scale proteome analyses of Saccharomyces cerevisiae and human embryonic stem cells, we identified 53,055 peptides in total, which was greater than by using CAD (38,293) or ETD (39,507) alone. In addition, the DT method also identified 7,422 phosphopeptides, compared to either 2,801 (CAD) or 5,874 (ETD) phosphopeptides.     

Analytical aspects of mass spectrometry and proteomics

Mass spectrometry plays an essential role in proteomics analysis and research. In recent years, it has been increasingly recognized that a key to proteomics using mass spectrometry relies not only on the instrument itself, but also on the analytical strategies and front-end sample-handling techniques. The advances of separations and mass spectrometry are having an increasing impact on the discovery of disease biomarkers and the understanding of cellular processes.     

Spatial proteomics of vesicular trafficking: coupling mass spectrometry and imaging approaches in membrane biology

In plants, membrane compartmentalization requires vesicle trafficking for communication among distinct organelles. Membrane proteins involved in vesicle trafficking are highly dynamic and can respond rapidly to changes in the environment and to cellular signals. Capturing their localization and dynamics is thus essential for understanding the mechanisms underlying vesicular trafficking pathways. Quantitative mass spectrometry and imaging approaches allow a system-wide dissection of the vesicular proteome, the characterization of ligand-receptor pairs and the determination of secretory, endocytic, recycling and vacuolar trafficking pathways. In this review, we highlight major proteomics and imaging methods employed to determine the location, distribution and abundance of proteins within given trafficking routes. We focus in particular on methodologies for the elucidation of vesicle protein dynamics and interactions and their connections to downstream signalling outputs. Finally, we assess their biological applications in exploring different cellular and subcellular processes.     

Intact protein mass spectrometry and top-down proteomics

American Society for Mass Spectrometry Sanibel meeting on top-down mass spectrometry

St Pete Beach, FL, USA, 24–27 January 2013

Top-down mass spectrometry involves analysis of intact proteins, typically using electrospray ionization, as multiple charging enhances dissociation and thus identification by comparison of precursor and product ion masses with protein sequence databases. Traditionally a low-throughput, precision technology performed on high-resolution Fourier-transform ion cyclotron resonance mass analyzers, top-down proteomics aims to increase throughput for whole proteome analysis while preserving the inherent value of an intact protein mass measurement. This years’ American Society for Mass Spectrometry Sanibel meeting brought together established scientists who have demonstrated the viability of the top-down approach and its applicability to virtually all segments of the proteome, mixing them with researchers from diverse areas and with the common interest of advancing top-down into the high-throughput proteomics mainstream. Advances in instrumentation including the orbitrap analyzer, ionization mechanisms, dissociation strategies and informatics, as well as a wide variety of applications, were discussed in depth, leading to the inescapable conclusion that the future for top-down is bright.     

生物质谱与蛋白质组学

蛋白质组学是后基因组学时代最受关注的研究领域之一,其核心的鉴定技术——生物质谱近年来在仪器设计以及鉴定通量、分辨率和灵敏度等各方面均有质的飞跃,促进了蛋白质表达谱作图、定量蛋白质组分析、亚细胞器蛋白质组作图、蛋白质翻译后修饰以及蛋白质相互作用等蛋白质组研究各个领域的飞速发展。本综述了生物质谱技术的最新进展,及其在蛋白质组学研究中的应用。     

A label-free mass spectrometry method for the quantification of protein isotypes

Successful quantitative mass spectrometry (MS) requires strategies to link the mass spectrometer response to the analyte abundance, with the response being dependent on more factors than just analyte abundance. Label-dependent strategies rely on the incorporation of an isotopically labeled internal standard into the sample. Current label-free strategies (performed without internal standards) are useful for analyzing samples that are unsuitable for isotopic labeling but are less accurate. Here we describe a label-free technique applicable to analysis of products from related genes (isotypes). This approach enables the invariant tryptic peptide sequences within the family to serve as “built-in” internal standards and the isotype-specific peptide sequences to report the amount of the various isotypes. A process of elimination segregates reliably trypsin-released standard and reporter peptides from unreliably released peptides. The specific MS response factors for these reporter and standard peptides can be determined using synthetic peptides. Analysis of HeLa tubulin digests revealed peptides from βI-, βII-, βIII-, βIVb-, and βV-tubulin, eight of which were suitable; along with five standard peptides for quantification of the β-tubulin isotypes. To show the utility of this method, we determined that βI-tubulin represented 77% and βIII-tubulin represented 3.2% of the total HeLa β-tubulin.     

Advanced nanoscale separations and mass spectrometry for sensitive high-throughput proteomics

Recent developments in combined separations with mass spectrometry for sensitive and high-throughput proteomic analyses are reviewed herein. These developments primarily involve high-efficiency (separation peak capacities of ~10³) nanoscale liquid chromatography (flow rates extending down to approximately 20 nl/min at optimal liquid mobile-phase separation linear velocities through narrow packed capillaries) in combination with advanced mass spectrometry and in particular, high-sensitivity and high-resolution Fourier transform ion cyclotron resonance mass spectrometry. Such approaches enable analysis of low nanogram level proteomic samples (i.e., nanoscale proteomics) with individual protein identification sensitivity at the low zeptomole level. The resultant protein measurement dynamic range can approach 10⁶ for nanogram-sized proteomic samples, while more abundant proteins can be detected from subpicogram-sized (total) proteome samples. These qualities provide the foundation for proteomics studies of single or small populations of cells. The instrumental robustness required for automation and providing high-quality routine performance nanoscale proteomic analyses is also discussed.     

Advanced nanoscale separations and mass spectrometry for sensitive high-throughput proteomics

Recent developments in combined separations with mass spectrometry for sensitive and high-throughput proteomic analyses are reviewed herein. These developments primarily involve high-efficiency (separation peak capacities of approximately 10(3)) nanoscale liquid chromatography (flow rates extending down to approximately 20 nl/min at optimal liquid mobile-phase separation linear velocities through narrow packed capillaries) in combination with advanced mass spectrometry and in particular, high-sensitivity and high-resolution Fourier transform ion cyclotron resonance mass spectrometry. Such approaches enable analysis of low nanogram level proteomic samples (i.e., nanoscale proteomics) with individual protein identification sensitivity at the low zeptomole level. The resultant protein measurement dynamic range can approach 10(6) for nanogram-sized proteomic samples, while more abundant proteins can be detected from subpicogram-sized (total) proteome samples. These qualities provide the foundation for proteomics studies of single or small populations of cells. The instrumental robustness required for automation and providing high-quality routine performance nanoscale proteomic analyses is also discussed.     

Spectral quality assessment for high-throughput tandem mass spectrometry proteomics

Current techniques in tandem mass spectrometric analyses of cellular protein contents often produce thousands to tens of thousands of spectra per experiment. This study introduces a new algorithm, named SPEQUAL, which is aimed at automated tandem mass spectral quality assessment. The quality of a given spectrum can be evaluated from three basic components: (i) charge state differentiation, (ii) total signal intensity, and (iii) signal-to-noise estimates. The differentiation between single and multiple precursor charge states (i) provides a binary score for a given spectrum. Components (ii) and (iii) provide partial scores which are subsequently summarized and multiplied by the first score. SPEQUAL was applied to over 10,000 data files derived from almost 3,000 tandem mass spectra, and the results (final cumulative scores) were manually verified. SPEQUAL's performance was determined to have high sensitivity and specificity and low error rates for both spectral quality estimates in general and precursor charge state differentiation in particular. Each of the partial scores is controlled by adjustable thresholds to fine-tune SPEQUAL's performance for different analysis pipelines and instrumentation. This spectral quality assessment tool is intended to act in an advisory role to the researcher, assisting in filtration of thousands of spectra typically produced by high throughput tandem mass spectrometric proteome analyses. Lastly, SPEQUAL was implemented as Java GUI-based and command-line-based interfaces freely available for both academic and industrial researchers.     

Performance of a genetic algorithm for mass spectrometry proteomics

Background  

Recently, mass spectrometry data have been mined using a genetic algorithm to produce discriminatory models that distinguish healthy individuals from those with cancer. This algorithm is the basis for claims of 100% sensitivity and specificity in two related publicly available datasets. To date, no detailed attempts have been made to explore the properties of this genetic algorithm within proteomic applications. Here the algorithm's performance on these datasets is evaluated relative to other methods.     

Bioinformatics in mass spectrometry data analysis for proteomics studies

Mass spectrometry is a technique widely employed for the identification and characterization of proteins. The role of bioinformatics is fundamental for the elaboration of mass spectrometry data due to the amount of data that this technique can produce. To process data efficiently, new software packages and algorithms are continuously being developed to improve protein identification and characterization in terms of high-throughput and statistical accuracy. However, many limitations exist concerning bioinformatics spectral data elaboration. This review aims to critically cover the recent and future developments of new bioinformatics approaches in mass spectrometry data analysis for proteomics studies.