W. M. Keck FT-ICR Mass Spectrometry Laboratory, Department of Chemistry, North Carolina State University, Raleigh, North Carolina 27695, USA.
Anal Chem. 2010 Oct 15;82(20):8696-702. doi: 10.1021/ac101978b.
Numerous experimental strategies exist for relative protein quantification, one of the primary objectives of mass spectrometry based proteomics analysis. These strategies mostly involve the incorporation of a stable isotope label via either metabolic incorporation in cell or tissue culture (¹⁵N/¹⁴N metabolic labeling, stable isotope labeling by amino acids in cell culture (SILAC)), chemical derivatization (ICAT, iTRAQ, TMT), or enzymatically catalyzed incorporation (¹⁸O labeling). Also, these techniques can be cost or time prohibitive or not amenable to the biological system of interest (i.e., metabolic labeling of clinical samples, most animals, or fungi). This is the case with the quantification of fungal proteomes, which often require auxotroph mutants to fully metabolically label. Alternatively, label-free strategies for protein quantification such as using integrated ion abundance and spectral counting have been demonstrated for quantification affording over 2 orders of magnitude of dynamic range which is comparable to metabolic labeling strategies. Direct comparisons of these quantitative techniques are largely lacking in the literature but are highly warranted in order to evaluate the capabilities, limitations, and analytical variability of available quantitative strategies. Here, we present the direct comparison of SILAC to label-free quantification by spectral counting of an identical set of data from the bottom-up proteomic analysis of human embryonic stem cells, which are readily able to be quantified using both strategies, finding that both strategies result in a similar number of protein identifications. We also discuss necessary constraints for accurate quantification using spectral counting and assess the potential of this label-free strategy as a viable alternative for quantitative proteomics.
存在许多用于相对蛋白质定量的实验策略，这是基于质谱的蛋白质组学分析的主要目标之一。这些策略主要涉及通过细胞或组织培养中的代谢掺入（¹⁵N/¹⁴N 代谢标记、稳定同位素标记的细胞培养氨基酸（SILAC））、化学衍生化（ICAT、iTRAQ、TMT）或酶促催化掺入（¹⁸O 标记）来掺入稳定同位素标记。此外，这些技术可能成本高昂或耗时较长，或者不适用于感兴趣的生物系统（例如，临床样本、大多数动物或真菌的代谢标记）。在真菌蛋白质组定量方面就是这种情况，通常需要营养缺陷型突变体才能进行完全代谢标记。或者，已经证明了用于蛋白质定量的无标记策略，例如使用集成离子丰度和谱计数，可提供超过 2 个数量级的动态范围，这与代谢标记策略相当。这些定量技术的直接比较在文献中基本上是缺乏的，但为了评估可用定量策略的能力、限制和分析可变性，这是非常有必要的。在这里，我们通过对人类胚胎干细胞的从头蛋白质组分析的相同数据集的 SILAC 与基于谱计数的无标记定量进行直接比较，发现这两种策略都可以对数据进行定量，且这两种策略都产生了相似数量的蛋白质鉴定。我们还讨论了基于谱计数进行准确定量的必要约束，并评估了这种无标记策略作为定量蛋白质组学的可行替代方案的潜力。
