Interferences and contaminants encountered in modern mass spectrometry 现代质谱中遇到的干扰和污染物
Bernd O. Keller ^(a,**){ }^{a, *}, Jie Sui ^(b){ }^{b}, Alex B. Young ^(c){ }^{c}, Randy M. Whittal ^(d){ }^{d} Bernd O. Keller ^(a,**){ }^{a, *} ， Jie Sui ^(b){ }^{b} ， Alex B. Young ^(c){ }^{c} ， Randy M. Whittal ^(d){ }^{d}^(a){ }^{\mathrm{a}} Department of Pathology and Laboratory Medicine, University of British Columbia, Child&Family Research Institute, Vancouver, British Columbia V5T 1T7, Canada 不列颠哥伦比亚大学儿童与家庭研究所病理学和检验医学 ^(a){ }^{\mathrm{a}} 系，不列颠哥伦比亚省温哥华 V5T 1T7，加拿大^("b "){ }^{\text {b }} Department of Chemistry, Queen's University, Kingston, Ontario K7L 3N6, Canada 女王大学化学 ^("b "){ }^{\text {b }} 系，安大略省金斯敦 K7L 3N6，加拿大^("c "){ }^{\text {c }} Department of Chemistry, University of Toronto, Toronto, Ontario M5S 3H6, Canada 多伦多大学化学 ^("c "){ }^{\text {c }} 系，多伦多，安大略省 M5S 3H6，加拿大^(d){ }^{\mathrm{d}} Department of Chemistry, University of Alberta, Edmonton, Alberta T6G 2G2, Canada 阿尔伯塔大学化学 ^(d){ }^{\mathrm{d}} 系，埃德蒙顿，艾伯塔省 T6G 2G2，加拿大
A R T I C L E I N F O
Article history: 文章历史：
Received 12 February 2008 收稿日期 2008-02-12
Received in revised form 14 April 2008 2008年4月14日以订正表格收到
Accepted 16 April 2008 2008年4月16日录用
Published on line 25 April 2008 2008年4月25日发表于第1行
With the invention of electrospray ionization and matrix-assisted laser desorption/ionization, scientists employing modern mass spectrometry naturally face new challenges with respect to background interferences and contaminants that might not play a significant role in traditional or other analytical techniques. Efforts to continuously minimize sample volumes and measurable concentrations increase the need to understand where these interferences come from, how they can be identified, and if they can be eliminated. Knowledge of identity enables their use as internal calibrants for accurate mass measurements. This review/tutorial summarizes current literature on reported contaminants and introduces a number of novel interferences that have been observed and identified in our laboratories over the past decade. These include both compounds of proteinaceous and non-proteinaceous nature. In the supplemental data a spreadsheet is provided that contains a searchable ion list of all compounds identified to date. 随着电喷雾电离和基质辅助激光解吸/电离的发明，采用现代质谱的科学家自然面临着背景干扰和污染物方面的新挑战，这些挑战在传统或其他分析技术中可能不会发挥重要作用。不断减少样品量和可测量浓度的努力增加了了解这些干扰的来源、如何识别它们以及是否可以消除它们的需求。对身份的了解使其能够用作精确质量测量的内部校准品。本综述/教程总结了有关报告污染物的当前文献，并介绍了过去十年在我们的实验室中观察到和识别的许多新的干扰。这些包括蛋白质和非蛋白质性质的化合物。在补充数据中，提供了一个电子表格，其中包含迄今为止已鉴定的所有化合物的可搜索离子列表。
Proteinaceous interferences or contaminants … 72 蛋白质干扰或污染物......72
2.1. Enzymes used in sample preparations for bioanalytical mass spectrometry … 72 2.1. 用于生物分析质谱样品制备的酶 ...72
2.2. Keratins and other abundant, involuntarily introduced proteins … 73 2.2. 角蛋白和其他丰富的、不由自主地引入的蛋白质......73
2.3. Instrument-induced peptide fragment interferences … 74 2.3. 仪器诱导的肽片段干扰 ...74
Non-proteinaceous interferences or contaminants … 74 非蛋白质干扰或污染物......74
3.1. Matrix clusters in MALDI MS … 74 3.1. MALDI MS 中的基质簇 ...74
3.2. Adducts, solvents and polymeric interferences … 75 3.2. 加合物、溶剂和聚合物干扰物......75
3.3. Plasticizers, additives and other interferences … 78 3.3. 增塑剂、添加剂和其他干扰物......78
4. Explanation of background ions spreadsheet … 78 4. 背景离子电子表格说明 ...78
5. Conclusions … 79 5. 结论......79
Note added in proof … 79 在证明中添加的注释 ...79
Acknowledgements … 79 确认。。。79
Appendix A. Supplementary data … 79 附录 A. 补充数据 ...79
References … 79 引用。。。79
1. Introduction 1. 简介
“Scheidekunst”-the ‘art’ to separate materials into their individual components is an old German word for alchemy and analytical chemistry related sciences [1] and its basic concepts are still valid today. The last hundred years have brought enormous advances in chromatographic and other separation methods in combination with a large variety of analyte detection technologies. Any separation and detection technique has the potential to inadvertently introduce new components or contaminants into the analytical system that must be evaluated and carefully considered. It is therefore no coincidence that Modern Analytical Chemistry takes advantage of ultrapure chemicals and reagents and ultraclean sample handling containers whenever possible to minimize any potential and unwanted background interference. In addition, all routine modern analytical methods following good laboratory practices (GLP) will include blank tests such as system-, solvent-, method-, matrix- and equipment blanks [2]. “Scheidekunst”——将材料分离成各个组成部分的“艺术”是一个古老的德语单词，指炼金术和分析化学相关科学 [1]，其基本概念在今天仍然有效。过去一百年来，色谱和其他分离方法与各种分析物检测技术相结合，取得了巨大进步。任何分离和检测技术都有可能无意中将新的组分或污染物引入分析系统，必须对其进行评估和仔细考虑。因此，现代分析化学尽可能利用超纯化学品和试剂以及超净样品处理容器来最大限度地减少任何潜在和不必要的背景干扰，这并非巧合。此外，遵循良好实验室规范（GLP）的所有常规现代分析方法都将包括空白测试，例如系统、溶剂、方法、基质和设备空白[2]。
With the introduction of novel ionization methods for modern mass spectrometric (MS) analysis, such as electrospray ionization (ESI) by Fenn et al. [3] and matrix-assisted laser desorption/ionization (MALDI) by Tanaka et al. [4] and independently by Karas and Hillenkamp [5], in the late eighties, scientists employing modern mass spectrometric tools face new challenges with respect to background ions that might not have played significant roles in traditional or other well-established routine analytical methodologies. The ongoing efforts to further miniaturize liquid chromatography (LC) methods [6], combinations of LC/MS [7-12], the development of capillary electrophoresis combined with MS (CE/MS) [13] including microfluidic chip-based mass spectrometry [14-17], make it easy to predict that knowledge of potential interferences and background ions will become increasingly important for successful future development of GLP-adhering methods in routine and research analytical methodologies. Miniaturization of sample transfer procedures and handling tools increases exponentially the surface to sample volume ratio and thus any interferences resulting from contaminated or background leaching surfaces will consequently also multiply. 随着现代质谱（MS）分析的新型电离方法的引入，例如 Fenn 等[3]的电喷雾电离（ESI）和 Tanaka 等[4]的基质辅助激光解吸/电离（MALDI）以及 Karas 和 Hillenkamp[5]的独立研究，在 80 年代后期，使用现代质谱工具的科学家们面临着背景离子的新挑战，这些离子在传统或其他成熟的常规中可能没有发挥重要作用分析方法。液相色谱（LC）方法[6]的小型化努力[6]、LC/MS 的组合[7-12]、毛细管电泳与 MS 相结合的开发（CE/MS）[13]，包括基于微流控芯片的质谱[14-17]，使得预测潜在干扰和背景离子的知识对于未来在常规和研究分析方法中成功开发 GLP 粘附方法将变得越来越重要。样品传输程序和处理工具的小型化使表面与样品体积的比呈指数级增长，因此，由污染或背景浸出表面引起的任何干扰也会因此成倍增加。
The main focus of this review/tutorial is the introduction and description of known and identified interfering compounds that have either been reported in the scientific literature or have been observed in our own laboratories over the past decade or so. Where deemed appropriate and possible, general technical advice is included on how to minimize impacts of the described interferences. 本综述/教程的主要重点是介绍和描述已知和已鉴定的干扰化合物，这些化合物要么在科学文献中报道过，要么在过去十年左右的时间里在我们自己的实验室中观察到。在认为适当和可能的情况下，包括关于如何尽量减少所描述干扰的影响的一般技术建议。
This report does not include or further discuss specific techniques or instrumentation that allow minimization or elimination of certain background interferences, such as ion mobility MS [18,19], high-field asymmetric waveform ion mobility spectrometry (FAIMS) [20], matrix-free laser desorption/ionization techniques [21] including desorption/ionization on silicon (DIOS) [22], desorption electrospray ionization (DESI) [23,24], or direct analysis in real time (DART) [25]. Sources and handling of random background noise, either of electrical [26-28] or chemical nature [29], noise reduction through special software application [30], interferences introduced through degradation or metabolism of analytes/drugs during analysis [31,32], co- or post-translational protein modifications [ 33,34 ], background ion scrubbing via specific reactions with dimethylsulfide [35,36], or any analyte-specific interferences are also beyond the scope of this work. We would like to refer interested readers in the above-mentioned topics to the respective cited literature and the references therein. 本报告不包括或进一步讨论允许最小化或消除某些背景干扰的特定技术或仪器，例如离子淌度 MS[18,19]、高场不对称波形离子淌度谱（FAIMS）[20]、无基质激光解吸/电离技术[21]，包括硅上解吸/电离（DIOS）[22]、解吸电喷雾电离（DESI）[23,24]或实时直接分析（DART）[25]。随机背景噪声的来源和处理，无论是电噪声[26-28]还是化学噪声[29]，通过特殊软件应用程序降低噪声[30]，分析物/药物在分析过程中降解或代谢引入的干扰[31,32]，共翻译或翻译后蛋白质修饰[33,34]，通过与二甲基硫化物的特定反应进行背景离子洗涤[35,36]，或任何分析物特异性干扰，也超出了这项工作的范围。我们想向对上述主题感兴趣的读者推荐相应的引用文献及其参考文献。
The supplemental data contains a spreadsheet (MicrosoftExcel) with a searchable compilation of all identified compounds to date. The list contains accurate mass-to-charge ratios for singly charged species and these can be exploited for calibration purposes in applications that require accurate mass measurements. 补充数据包含一个电子表格 （MicrosoftExcel），其中包含迄今为止所有已识别化合物的可搜索汇编。该列表包含单电荷物质的精确质荷比，这些质荷比可用于需要精确质量测量的应用中的校准目的。
2. Proteinaceous interferences or contaminants 2. 蛋白质干扰或污染物
One of modern mass spectrometry’s great impacts is on protein analysis and characterization. MS related techniques are now the preferred and well-established methods for protein identification [37] and quantification [38]. The complexity of biological samples requires extensive purification and separation methodologies, not only to remove non-proteinaceous components but also to address present proteins that are not of interest [39,40]. Here we will focus on potentially interfering proteins that are not indigenous to the original samples and are either involuntarily introduced into the sample (e.g. keratins) or deliberately added as enzymatic reagents for example in bottom up sample preparation for subsequent mass spectrometric analysis. 现代质谱法对蛋白质分析和表征的重大影响之一是。MS 相关技术现在是蛋白质鉴定[37]和定量[38]的首选和成熟方法。生物样品的复杂性需要广泛的纯化和分离方法，不仅要去除非蛋白质成分，还要解决现有不感兴趣的蛋白质[39,40]。在这里，我们将重点关注非原始样品固有的潜在干扰蛋白，这些蛋白质要么不由自主地引入样品中（例如角蛋白），要么被故意添加为酶试剂，例如在自下而上的样品制备中，用于后续质谱分析。
2.1. Enzymes used in sample preparations for bioanalytical mass spectrometry 2.1. 用于生物分析质谱样品制备的酶
A typical bottom up approach of protein sample preparation for subsequent mass spectrometric analysis involves enzymatic digestion of the protein of interest followed by 用于后续质谱分析的蛋白质样品制备的典型自下而上方法包括对目标蛋白质进行酶消化，然后进行
Table 1 - List of some enzymes ^("a "){ }^{\text {a }} commonly used in bottom up sample preparations for MS analysis 表 1 - 自下而上样品制备中 ^("a "){ }^{\text {a }} 常用的一些酶列表，用于 MS 分析
Low or broad cleavage specificity, preferred hydrophobic residues F \\\backslash; L \\\backslash; E \\\backslash 低或宽的切割特异性，优选疏水残基 F \\\backslash ;L \\\backslash ;E \\\backslash
^(a){ }^{\mathrm{a}} Adopted from the Proteolytic Enzymes Index at www.sigmaaldrich.com.
^("b "){ }^{\text {b }} Accessible at www.expasy.org (Expert Protein Analysis System, ExPASy).
^(a) Adopted from the Proteolytic Enzymes Index at www.sigmaaldrich.com.
^("b ") Accessible at www.expasy.org (Expert Protein Analysis System, ExPASy).| ${ }^{\mathrm{a}}$ Adopted from the Proteolytic Enzymes Index at www.sigmaaldrich.com. |
| :--- |
| ${ }^{\text {b }}$ Accessible at www.expasy.org (Expert Protein Analysis System, ExPASy). |
peptide mapping and/or MS/MS fragmentation of gas-phase separated peptide ions [37]. In an ideal case the sample protein is in large excess so that potential interferences from the added enzyme are minimal. However, one of modern mass spectrometry’s great successes is the analysis of often low nanogram amounts of proteins separated and purified from complex mixtures with chromatographic or gel electrophoresis techniques. In these cases enzyme-to-protein ratios are often reversed so that potential interferences from enzyme autolysis products become significant and need to be addressed. Bovine and porcine trypsin are the most commonly employed enzymes in MS protein analysis protocols but “difficult” proteins, such as membrane proteins with few or non-accessible tryptic cleavage sites, might require other enzymes for sufficient digestion. Although, autolysis products of trypsin have been described in the literature [ 41,42 ], due to variability of digestion results under different experimental conditions, blank testing of enzyme autolysis remains advisable. To minimize interferences from peptide artifacts, either from enzymes or other sources, it is necessary to optimize digestion conditions and employ proteolytic enzymes of high purity [43]. The latter can be challenging; purification of enzymes requires techniques that do not affect their activity [44]. Table 1 summarizes the most commonly employed enzymes, including their specific cleavage sites and their online database accession number (SwissProt database). In cases where sequential multienzymatic digestions are necessary, for example in experiments where maximum protein sequence coverage is required [45], the complexity of potential interference will naturally increase and sophisticated and adequate blank test experiments are required. 气相分离肽离子的肽图分析和/或 MS/MS 碎裂[37]。在理想情况下，样品蛋白质过量，因此添加的酶的潜在干扰最小。然而，现代质谱法的巨大成功之一是使用色谱或凝胶电泳技术分析从复杂混合物中分离和纯化的蛋白质通常为低纳克量。在这些情况下，酶与蛋白质的比例通常会被颠倒，因此酶自溶产物的潜在干扰变得显着并需要解决。牛胰蛋白酶和猪胰蛋白酶是 MS 蛋白分析方案中最常用的酶，但“困难”蛋白质，例如胰蛋白酶切割位点很少或无法接近的膜蛋白，可能需要其他酶才能充分消化。尽管文献中已经描述了胰蛋白酶的自溶产物[ 41\u201242 ]，但由于不同实验条件下消化结果的差异，酶自溶的空白测试仍然是可取的。为了尽量减少酶或其他来源的肽伪影的干扰，有必要优化消化条件并采用高纯度的蛋白水解酶[43]。后者可能具有挑战性;酶的纯化需要不影响其活性的技术[44]。表 1 总结了最常用的酶，包括它们的特定切割位点和在线数据库登录号（SwissProt 数据库）。 在需要顺序多酶消化的情况下，例如在需要最大蛋白质序列覆盖率的实验中[45]，潜在干扰的复杂性自然会增加，并且需要复杂和充分的空白测试实验。
New promising methods and technologies have recently evolved that avoid the use of enzymes in sample preparations for mass spectrometric characterization of proteins. These include microwave-assisted acid hydrolysis for controlled protein digestion [46], or top-down MS analysis of whole proteins [47]. However due to the wide acceptance of the robust enzymatic digestion methods and initial challenges with the new approaches, it is unlikely that the use of enzymes in mass spectrometric sample preparation protocols will be significantly replaced in the near future [48]. 最近发展出新的有前途的方法和技术，避免在样品制备中使用酶来表征蛋白质的质谱。这些方法包括用于受控蛋白质消化的微波辅助酸水解[46]，或全蛋白的自上而下的 MS 分析[47]。然而，由于稳健的酶解方法被广泛接受，以及新方法的最初挑战，酶在质谱样品制备方案中的使用不太可能在不久的将来被显着取代[48]。
2.2. Keratins and other abundant, involuntarily introduced proteins 2.2. 角蛋白和其他丰富的、不由自主引入的蛋白质
Keratins are ubiquitous proteins stemming predominantly from skin cells and are commonly found in house-hold and laboratory dust [49,50] and have the potential to contaminate biological samples if appropriate care and precautions are not taken [51]. Due to their proteinaceous nature, contaminating keratins will inevitably also undergo enzymatic digestion in respective bottom up sample preparations for mass spectrometry, and the resulting peptides will interfere in the analysis of the proteins of interest. The less abundant the proteins of interest, the greater the possibility that keratins will interfere with analysis. Necessary measures to avoid keratin interference will thus depend on the investigated samples and can range from simply wearing gloves, to working in laminar flow hoods or to working in special clean rooms. An excellent guideline on how and when to avoid keratin interferences has been developed by Biringer [51]. In 1999, Mann and co-workers presented a comprehensive list with respective MS/MS data of common peptide contaminants [41] including keratins, and these contaminants are contained in our supplemental spreadsheet. One could expect that most keratins found in dust are of human origin and this is most likely true for the majority of laboratories, however if lab animal facilities are included or nearby or rodent infestation has occurred, respective keratins from the species in question should also be expected. Another prerogative in prudent lab practices is to be prepared and vigilant; during a major project investigating the mouse endoplasmic reticulum proteome [52], we started identifying a large number of highly abundant sheep keratins at one point. As it turned out, a lab member had started wearing a wool sweater in the collaborator’s sample preparation laboratory, initiated by a sudden outside temperature drop [53]. 角蛋白是一种普遍存在的蛋白质，主要来源于皮肤细胞，常见于家庭和实验室灰尘中[49,50]，如果不采取适当的护理和预防措施，有可能污染生物样品[51]。由于其蛋白质性质，污染的角蛋白在质谱的各自自下而上的样品制备中也不可避免地会进行酶消化，所得肽会干扰目标蛋白质的分析。感兴趣的蛋白质含量越低，角蛋白干扰分析的可能性就越大。因此，避免角蛋白干扰的必要措施将取决于所研究的样品，范围从简单地戴手套到在层流罩中工作或在特殊的洁净室中工作。Biringer[51]制定了关于如何以及何时避免角蛋白干扰的优秀指南。1999 年，Mann 及其同事提交了一份综合清单，其中包含包括角蛋白在内的常见肽污染物[41]的 MS/MS 数据，这些污染物包含在我们的补充电子表格中。人们可以预期，在灰尘中发现的大多数角蛋白都是人类来源的，这很可能对大多数实验室来说都是正确的，但是，如果包括实验动物设施或附近或发生了啮齿动物侵扰，则还应该预期来自相关物种的相应角蛋白。审慎实验室实践中的另一个特权是做好准备和保持警惕;在一个研究小鼠内质网蛋白质组的重大项目中[52]，我们一度开始鉴定出大量高丰度的绵羊角蛋白。 事实证明，一名实验室成员在合作者的样品制备实验室中开始穿着羊毛衫，这是由于室外温度突然下降而引发的[53]。
A number of other proteins can be involuntarily introduced into biological samples. Among these are bovine serum albumin (BSA) which is commonly employed in immunoassays and other techniques as a reagent to block non-specific binding sites on surfaces [54], or Protein A and G which are used as specific binding partners for immunoglobulins (IgG) in 许多其他蛋白质可以不由自主地引入生物样品中。其中包括牛血清白蛋白（BSA），它通常用于免疫测定和其他技术，作为阻断表面非特异性结合位点的试剂[54]，或蛋白 A 和 G，用作免疫球蛋白（immunoglobulins， IgG）的特异性结合伙伴。