Liquid Chromatography-Mass Spectrometry-based Quantitative Proteomics

Fang Xie, Tao Liu, Wei-Jun Qian, Vladislav A. Petyuk, Richard D. Smith
2011 Journal of Biological Chemistry  
LC-MS-based quantitative proteomics has become increasingly applied to a wide range of biological applications due to growing capabilities for broad proteome coverage and good accuracy and precision in quantification. Herein, we review the current LC-MS-based quantification methods with respect to their advantages and limitations and highlight their potential applications. LC-MS-based quantitative proteomic approaches have become increasingly popular over the past decade (1-7). In general,
more » ... very-based proteomic efforts lend themselves to global analyses whereby a broad survey of the proteome is performed across various samples, and the quantitative differences among them are estimated. In discovery-based efforts, the breadth of analysis is emphasized more than the precision and accuracy of quantification. For studies in which these qualities are crucial such as verification efforts, the tactic switches to sensitive, precise, and accurate analysis of a few targeted proteins in relatively large set of samples and internal standards are often used. Fig. 1 illustrates an LC-MS-based global proteomic workflow in which proteins are converted into peptides for identification and quantification (i.e. "bottom-up" proteomics). Typically, methods are applied in conjunction with enzymatic digestion of proteins and subsequent measurement of one or more peptides from each protein that serve as effective measurement surrogates. We note that direct measurement of intact proteins (i.e. "top-down" proteomics) is another analytical option but is beyond the scope of this minireview and therefore not discussed herein. In global analyses, relative quantification of peptides usually involves either label-free or stable isotope labeling techniques (1) to discern differences in protein abundances among different biological conditions, and results are often expressed as "-fold changes." Overall, label-free approaches have wider dynamic range and broader proteome coverage, whereas stable isotope labeling approaches offer higher quantification precision and accuracy (8). Another common approach is absolute quantification, which determines the exact amount or concentration of a peptide/protein in a given sample and requires the use of an appropriate "internal" standard. All of these approaches have considerably different pros and cons that must be weighed before deciding which one is best for a specific course of study. Challenges affecting quantification in a bottom-up proteomic workflow stem from the wide range of peptide and protein physicochemical properties that give rise to large differences in MS responses (8). Sample handling, digestion efficiency, and separation also can have an impact on results. As such, relative peptide intensities may not directly reflect the relative abundances of different proteins. A major factor that influences LC-MS-based quantification via electrospray ionization is ion suppression (9). Peptide intensity depends on the quantity of the peptide being ionized as well as on ionization efficiency and, under some conditions, on the properties of coeluting peptides. The use of lower flow rates (e.g. Ͻ100 nl/min) (9, 10) or internal standards (11, 12) can help alleviate ion suppression. Other issues in LC-MS-based quantification include the separation peak capacity and reproducibility of the chromatography and the mass measurement accuracy and resolving power of the mass spectrometer. Significant technological advances such as the development and commercialization of ultra-performance LC and high-mass accuracy/resolution mass spectrometers have substantially overcome these issues, making LC-MS-based quantification more reliable and accessible to biologists. Basically, there is no recognized "one-size-fits-all" method that fulfills every quantitative need, and available options for quantification can make it difficult for an investigator to choose the most appropriate approach to answer particular biological questions. This minireview presents the advantages and limitations of commonly used LC-MS-based protein quantification approaches and provides guidelines for researchers who may not be familiar with but would like to benefit from quantitative proteomic measurements. Label-free Quantification Straightforward and inexpensive, label-free quantification is being increasingly applied to proteomic measurements. Without the need to modify peptides/proteins with stable isotopecontaining compounds or to add heavy isotope-labeled internal standards to the sample, label-free approaches require minimal manipulation of the sample and can be used on any type of biological materials. Conceptually, label-free quantification allows for the comparison of an unlimited number of samples; however, each sample has to be analyzed individually (no sample multiplexing). This type of approach usually offers wide dynamic range, which is especially advantageous when rela-
doi:10.1074/jbc.r110.199703 pmid:21632532 pmcid:PMC3138289 fatcat:i6u7yfdugbfwdeycakftmizw6e