Sample Preparation

*Mass Spectrometry in Life Sciences and Clinical Laboratory*

[21] Sanders NL, Sokol E, Perry RH, Huang G, Noll RJ, Duncan JS, et al. Hand-held mass spectrometer for environmentally relevant analytes using a variety of sampling and ionization methods. Eur J Mass Spectrom (Chichester). 2010;16(1):11-20.

[22] Gao L, Sugiarto A, Harper JD, Cooks RG, Ouyang Z. Design and characterization of a multisource hand-held tandem mass spectrometer. Anal Chem. 2008;80(19):7198-205.

[23] Keil A, Talaty N, Janfelt C, Noll RJ, Gao L, Ouyang Z, et al. Ambient mass spectrometry with a handheld mass spectrometer at high pressure. Anal

Chem. 2007;79(20):7734-9.

[14] Malla TR, Tumber A, John T, Brewitz L, Strain-Damerell C, Owen CD, et al. Mass spectrometry reveals potential of β-lactams as SARS-CoV-2 M(pro) inhibitors. Chemical communications (Cambridge, England).

2021;57(12):1430-3.

2020;92(21):14314-8.

[15] Sørensen LK, Petersen A, Granfeldt A, Simonsen U, Hasselstrøm JB. A validated UHPLC-MS/MS method for rapid determination of senicapoc in plasma samples. Journal of pharmaceutical and biomedical analysis. 2021;197:113956.

[16] Meisenbichler C, Kluibenschedl F, Müller T. A 3-in-1 Hand-Held Ambient Mass Spectrometry Interface for Identification and 2D Localization of Chemicals on Surfaces. Anal Chem.

[17] Wang X, Zhou X, Ouyang Z. Direct Analysis of Nonvolatile Chemical Compounds on Surfaces Using a Hand-

Held Mass Spectrometer with Synchronized Discharge Ionization

[18] Hendricks PI, Dalgleish JK,

and performance. Anal Chem.

2014;86(6):2900-8.

Shelley JT, Kirleis MA, McNicholas MT, Li L, et al. Autonomous in situ analysis and real-time chemical detection using a backpack miniature mass spectrometer: concept, instrumentation development,

[19] Chen TC, Ouyang Z. Synchronized discharge ionization for analysis of volatile organic compounds using a hand-held ion trap mass spectrometer. Anal Chem. 2013;85(3):1767-72.

[20] Dunn JD, Gryniewicz-Ruzicka CM,

Buhse LF. Using a portable ion mobility

supplements for sibutramine. Journal of pharmaceutical and biomedical analysis.

Kauffman JF, Westenberger BJ,

spectrometer to screen dietary

2011;54(3):469-74.

Function. Anal Chem. 2016;88(1):826-31.

**8**

**11**

**Chapter 2**

**Abstract**

**1. Introduction**

as a target for medical treatment.

Trends in Sample Preparation for

Sample preparation is a key step in proteomics, however there is no consensus in the community about the standard method for preparation of proteins from clinical samples like tissues or biofluids. In this chapter, we will discuss some important steps in sample preparation used for bottom-up proteome profiling with mass spectrometry (MS). Specifically, tissues, which are an important source of biological information, are of interest because of their availability. Tissues are most often stored as fresh frozen (FF) or formalin-fixed paraffin-embedded (FFPE). While FF tissues are more readily available, paraffin embedding has historically been routinely used for tissue preservation. However, formaldehyde induced crosslinks during FFPE tissue preservation present a challenge to the protocols used for protein retrieval. Moreover, in our view, an important aspect to consider is also the amount of material available at the start of a protocol since this is directly related to the choice of protocol in order to minimize sample loss and maximize detection of peptides by MS. This "MS sensitivity" is of special importance when working with patient samples that are unique and often available in limited amounts making optimization of methods to analyze the proteins therein important given that their

molecular information can be used in a patients' diagnosis and treatment.

**Keywords:** sample preparation, tissue, digestion, mass spectrometry, proteomics

Proteomics is an important tool in the study of human biological material with the aim to extract knowledge that can improve a patients' treatment outcomes. Molecular information obtained from patient samples can be complementary to pathological observations all with the goal of faster and more accurate diagnosis, and subsequent treatment. Molecular analysis of tissue by proteomics can lead to disease classification and reveal underlying disease pathways that can further serve

Sample size and origin is an important aspect in sample preparation. Today, there are numerous sample preparation procedures existing which aim to improve sensitivity of detection or protein recovery from a sample. Release of proteins from native or artificial material is a crucial step in sample preparation and to improve protein recovery there are different additives such as detergents, chaotropes, buffers and salts added during the sample preparation that must be considered. Moreover, targeting special groups of proteins (e.g. membrane proteins), which are involved in key cellular functions and may be a target of pharmaceutical treatment, often represent a

*Jakub Faktor, David R. Goodlett and Irena Dapic*

Proteome Analysis
