Robert Ahrends Cristina Coman

Omics fields aim to comprehensively asses the components of biological systems and have identified so far tens of thousands of molecules, over an extensive abundance range. But how do we search for the needle in this molecular haystack? Articles assembled in this Special Issue on Targeted Omics describe some experimental and computational means needed to search for specific biomolecules in the haystack.

Asking the right questions is one of the pillars of hypothesis‐driven research. Yet, the answer might require focusing the analysis on a few specific biomolecules. As looking for a needle in a haystack, targeted mass spectrometry (MS), developed in the late 1970s,^[1, 2^] can help by monitoring the right set of biomolecules with high sensitivity, reproducibility, and quantitative accuracy. Traditionally, triple quadrupole and quadrupole/linear ion trap mass spectrometers were utilized to deplete molecules which are not of interest and to enrich the ones which are. Nevertheless, due to the potential of high‐resolution instruments, acquisition modes such as parallel reaction monitoring (PRM)^[3, 4^] or targeted single ion monitoring^[5^] are complementing nowadays selected reaction monitoring (SRM) on low‐resolution instruments.^[6^] Thereby, the targeted acquisition mode per se is not quantitative; however, in conjunction with authentic standards (isotope‐coded molecules) or surrogate standards (endogenous non‐occurring but structurally very similar) and the utilization of calibration curves, the concentration of the targeted molecules can be determined.^[7-10^]

Over the past decades, the use of quadrupole‐based instruments for targeted applications has escalated as methodological advances have made the technology more available. Efforts have been made on improving both the selectivity and the throughput to allow comparisons of a specific subset of molecules (e.g., biomarkers, low abundant molecules, pathways) such as those found usually in clinical, biochemistry, or industrial research settings. Instrumental developments led to the scanning speed of triple quadrupole instruments to increase remarkably over the last 10 years, facilitating today the measurement of more than 600 transitions per second and over 200 protein targets on a modern triple quadrupole instrument.^[11, 12^] Using the ion‐funnel technology to focus the entering ion beam, further improved the sensitivity of targeted assays.^[13^] However, triple quadrupole‐based targeted proteomics is still a low‐resolution approach to analyze peptides. Here, the selected ions cannot be accumulated during acquisitions limiting the specificity and sensitivity of this type of mass analyzer. Recently a high‐resolution PRM workflow was introduced. The workflow addresses this challenge and demonstrates the benefits of a quadrupole orbitrap mass analyzer in the analysis of very low abundant protein signaling molecules, which were not detectable in a common SRM setup.^[14^] These improvements are obviously just a few of those that contribute significantly to various research applications by boosting the sensitivity, reproducibility, and identification accuracy. Nevertheless, with such a remarkable molecular diversity and more and more questions to answer, targeted analyses are under a constant need for tools and strategies to look for the needle in the haystack. This special technical issue gives a detailed perspective about latest tools used in the field and potential application schemes for your targeted work.

A major bottleneck in the development of a robust SRM assay is raised by the physicochemical properties of the tryptic peptides. Not all peptides are equally ionizable and therefore detectable. In addition, they have to be carefully chosen to ensure that they uniquely represent the protein of interest. To this, Hentschel et al.^[15^] chose the peptides from a simple targeted assay for metabolic pathways & signaling (STAMPS) spectral library and combined the workflow with isotope‐coded internal standard‐based and sample prefractionation assays to monitor the metabolic key pathways during adipogenesis. The SRM approach enabled the detection and quantification also of very low abundant key proteins, covering a broad range of metabolic pathways in a time‐resolved manner.

Another way to tackle the challenge of quantifying very low abundant proteins is described by Camparini et al.^[16^] In order to differentiate between the circulating levels of two proteins which share 90% amino acid sequence identity (growth differentiation factor 11 [GDF11] and myostatin [GDF8]), the authors combined a PRM assay with immunoprecipitation. This strategy allowed for the quantification in the low ng per mL range of GDF11 and opened new venues for the role of gender and pathological conditions on the circulating levels of these two proteins.

However, targeted analysis still suffers due to the inevitable tradeoff between the instrument performance and the number of target molecules. To mitigate this tradeoff a Triggered by Offset, Multiplexed, Accurate mass, High resolution, and Absolute Quantitation (TOMAHAQ) approach^[17^] was developed. TOMAHAQ employs high‐resolution mass spectrometry in order to be able to combine tandem mass tag workflows and targeted proteomics in a promising trend which enables the quantification of up to ten samples in parallel. The approach enhances throughput and comparability across samples. However, in this mass tag‐based approach interferences derived from the tag itself can be observed. Fang et al.^[18^] are evaluating this interference and introduce a post‐acquisition data correction procedure and provide additional technical improvements to overcome interference issues.

Aiming to maintain the robust quantitation offered by targeted assays but on the scale of hundreds of lipids, Calderon et al.^[19^] combine a targeted data processing approach on untargeted lipidomics data generated by data independent acquisition (DIA) with sequential window acquisition of all theoretical fragment ion mass spectra (SWATH) in order to investigate betulin‐induced changes in keratinocytes. Therefore, the targeted identification strategy combines information from precursor and product ions obtained from analyses in both polarities under same chromatographic conditions and from elution patterns in reversed‐phase chromatography of each lipid class. This targeted data processing is including MS/MS information, retention time, and retention time dependencies of related lipid species which highly facilities the identification and rejection of false positively identified lipids.

However, the benefits of targeted MS analysis have not only been applied to LC‐MS/MS workflows. Direct infusion‐based shotgun lipidomics has been exploiting this approach for more than two decades. In their article, Hu et al.^[20^] summarize the general limitations the classical shotgun approach suffers from and review strategies (such as modifier addition, chemical derivatization, charge feature utilization) that have been employed to alleviate these limitations (high resolution and multidimensional mass spectrometry‐based shotgun lipidomics). Through these enhancements, lipid species from more than 50 lipid classes can be identified and quantified in a relatively high‐throughput manner.

Finally, the technical special issue presented here not only gives a brief overview about current targeted application in proteomics and lipidomics but also offers a critical perspective on techniques already provided and delivers bioinformatics tools generally applicable for quantitative assays. CalibraCurve^[21^] is such a tool that enables an automated batch‐mode determination of dynamic linear ranges and quantification limits for both targeted proteomics and lipidomics assays. CalibraCurve employs a multitude of measures to assess precision and trueness of the calibration. Thereby, accuracy measures and the provided graphs offer a detailed access to assess the quality of fit, making the tool very useful to foster the usage of calibration curves in any kind of targeted experiment.

罗伯特·阿伦兹·克里斯蒂娜·科曼

Omics领域旨在全面评估生物系统的组成部分，迄今已在广泛的丰度范围内鉴定出数万个分子。但是，我们如何在这个分子堆中寻找针头呢？本期《针对靶组学的特刊》中的文章描述了在干草堆中搜索特定生物分子所需的一些实验和计算方法。

提出正确的问题是假设驱动研究的支柱之一。但是，答案可能需要将分析重点放在一些特定的生物分子上。当在大海捞针中寻找目标时，目标质谱（MS）于1970年代后期开发，^[ 1、2 ^]可以通过以高灵敏度，可重复性和定量准确性监测正确的生物分子来提供帮助。传统上，使用三重四极杆和四极杆/线性离子阱质谱仪来耗尽不需要的分子并富集感兴趣的分子。然而，由于高分辨率仪器的潜力，采集模式例如平行反应监测（PRM）^[ 3，4 ^]或目标单离子监测^[ 5 ^]现在是对低分辨率仪器上选择的反应监测（SRM）的补充。^[ 6 ^]因此，目标获取模式本身不是定量的；但是，结合真实的标准品（同位素编码的分子）或替代标准品（内源性非存在但结构上非常相似）和利用校准曲线，可以确定目标分子的浓度。^[ 7-10 ^]

在过去的几十年中，随着方法学的进步使该技术更加可用，针对目标应用的基于四极杆的仪器的使用也在不断升级。已经在提高选择性和通量上做出了努力，以允许比较分子的特定子集（例如，生物标志物，低丰度分子，途径），例如通常在临床，生物化学或工业研究环境中发现的那些。仪器的发展导致三重四极杆仪器的扫描速度在过去10年中得到了显着提高，今天，这促进了现代三重四极杆仪器每秒600多个离子对和200多个蛋白质靶标的测量。^[ 11，12 ^]使用离子漏斗技术聚焦入射的离子束，进一步提高了目标测定的灵敏度。^[ 13 ^]然而，基于三重四极杆的靶向蛋白质组学仍然是分析肽段的低分辨率方法。在此，所选离子不能在采集过程中累积，从而限制了这种质量分析仪的特异性和灵敏度。最近，引入了高分辨率PRM工作流程。该工作流程解决了这一挑战，并证明了四极轨道质谱仪在分析非常低的丰富蛋白质信号分子中的优势，而这在普通SRM设置中是无法检测到的。^[ 14 ^]这些改进显然只是通过提高灵敏度，可重复性和识别准确性而对各种研究应用做出重大贡献的一小部分。然而，由于具有如此杰出的分子多样性以及越来越多的问题需要回答，针对性分析一直在寻找工具和策略来寻找大海捞针。该特殊技术问题详细介绍了该领域中使用的最新工具以及针对您的目标工作的潜在应用方案。

胰蛋白酶肽的理化性质提高了开发强大的SRM分析方法的主要瓶颈。并非所有的肽都能同等地离子化，因此可以被检测到。此外，必须仔细选择它们，以确保它们唯一代表目标蛋白。为此，Hentschel等。^[ 15 ^]从用于代谢途径和信号传导（STAMPS）光谱库的简单靶向分析中选择了肽段，并将工作流程与基于同位素编码的内标和样品预分级分析相结合，以监测脂肪形成过程中的代谢关键途径。SRM方法还可以检测和定量非常低的丰富关键蛋白，并以时间分辨的方式涵盖了广泛的代谢途径。

Camparini等人描述了解决定量非常低的丰富蛋白质的挑战的另一种方法。^[ 16 ^]为了区分具有90％氨基酸序列同一性的两种蛋白质（生长分化因子11 [GDF11]和肌生长抑制素[GDF8]）的循环水平，作者将PRM分析与免疫沉淀相结合。该策略允许在低ng / mL的GDF11范围内进行定量，并为性别和病理状况在这两种蛋白的循环水平上的作用开辟了新的场所。

然而，由于仪器性能和目标分子数量之间不可避免的折衷，目标分析仍然受到影响。为了减轻这种折衷，开发了一种由偏移，多路复用，精确质量，高分辨率和绝对定量（TOMAHAQ）触发的方法^[ 17 ^]。TOMAHAQ采用高分辨率质谱仪，以便能够将串联质谱标签工作流程与目标蛋白质组学结合在一起，并成为一个有希望的趋势，该趋势使得能够并行定量多达十个样品。该方法提高了样品的通量和可比性。但是，在这种基于标签的大规模方法中，可以观察到来自标签本身的干扰。方等。^[ 18 ^] 正在评估这种干扰，并介绍了采集后数据校正程序，并提供了其他技术改进来克服干扰问题。

为了维持靶向分析所提供的可靠定量，但要以数百种脂质的规模进行分析，Calderon等人。^[ 19 ^]将针对数据独立采集（DIA）生成的非目标脂质组学数据的目标数据处理方法与所有理论碎片离子质谱（SWATH）的顺序窗口采集相结合，以研究由白蛋白引起的角质形成细胞变化。因此，有针对性的鉴定策略结合了前体离子和产物离子的信息，前者离子和产物离子是通过在相同色谱条件下在两种极性下进行的分析以及每种脂质类别的反相色谱中的洗脱模式而获得的。这种有针对性的数据处理包括MS / MS信息，相关脂质种类的保留时间和保留时间相关性，这极大地促进了对假阳性的脂质的鉴定和剔除。

但是，目标质谱分析的好处不仅适用于LC-MS / MS工作流程。基于直接输注的shot弹枪脂质组学已经使用这种方法已有二十多年了。在Hu等人的文章中。^[ 20 ^]总结了传统shot弹枪方法受到的一般限制，并回顾了缓解这些限制（高分辨率和基于多维质谱的shot弹枪脂质组学）的策略（例如改性剂添加，化学衍生化，电荷特征利用）。通过这些增强功能，可以以相对较高的通量方式识别和量化来自50多个脂质类别的脂质种类。

最后，此处介绍的技术专刊不仅简要概述了蛋白质组学和脂质组学的当前目标应用，而且还对已提供的技术提供了重要的看法，并提供了通常可用于定量测定的生物信息学工具。CalibraCurve ^[ 21 ^]是一种这样的工具，它可以针对目标蛋白质组学和脂质组学测定方法，自动对动态线性范围和定量限进行批处理模式确定。CalibraCurve采用多种措施来评估校准的准确性和真实性。因此，准确度测量和提供的图形提供了评估拟合质量的详细途径，从而使该工具对于在任何类型的目标实验中促进使用校准曲线非常有用。