Protein quantification by MS

MS-based proteomics has rapidly evolved over the past years from a qualitative to a more quantitative approach ²³⁹. Although LC-MS is inherently a quantitative platform, the signal is subjected to variations. These variations are principally due to changes in the instrument performance, including variations of the injection volumes and degradation of the chromatographic column performance regarding the LC part; and the contamination or drift in the calibration regarding the MS system. In addition, the competition for the ionization in the ion source can suppress, or sometimes enhance, the signal of an ion species. Several strategies can be used for protein quantification. These can be broadly divided in label-based and label-free approaches.

5.1.2.1. Label-based approaches

An efficient approach to control for the variations in the sample preparation and/or LC-MS analysis consists in the incorporation ofamino acids labeled with stable isotopes (¹³C, ¹⁵N and/or ¹⁸O) into internal standards. The isotope-labeled peptides, often called heavy peptides, display the same sequences and similar physico-chemical characteristics to that of their endogenous peptides, also called light peptides, but are distinguishable by MS due to their mass increase ²⁴⁰. Equal amounts of labeled internal standard are added to all samples to be analyzed and, as each heavy and endogenous peptide pair displays the same chromatographic behavior, ionization efficiency, and fragmentation patterns, the MS signal

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of each endogenous peptide can be normalized by the signal of its isotopic labeled version to control for variability factors. Furthermore, the labeled internal standards should be spiked as early as possible during the sample preparation procedure in order to control the maximum number of steps of the sample processing and decrease the technical variability

239. The addition of isotope labeled standards not only enables a more accurate relative or absolute quantification but also provides an extra step on the confidence of the peptide identification due to the co-elution of the endogenous peptide and the internal standard (used as the reference) and the possibility to perform spectral matching ²⁴¹.

Several strategies are employed to introduce labeled internal standards, including the use of isotopically labeled proteins, synthetic peptides or isotopically labeled derivatization reagents.

! Isotopically labeled proteins

Full-length isotope-labeled proteins are the ideal standard for quantitative proteomics ^242,243. In contrast to peptide standards, adding isotope-labeled proteins in early steps of the sample preparation workflow enables to control for variations that may occur during proteolysis and pre-fractionation steps.

The chemical synthesis of proteins is almost impossible due to their size (it is difficult to synthesize over 30 amino acids) and the challenge to reproduce the specific folding and tridimensional structure. Metabolic incorporation by stable isotope labeling by amino acids in cell culture (SILAC) is a feasible method for the production of isotope-labeled proteins. In another biological condition that is not labeled and they are quantitatively compared. With this approach, the relative quantification of the complete proteome between two states is possible. However, since SILAC requires complete metabolic labeling of proteomes, it is applicable only to cultured cells or, at most, to small organisms like mice ²⁴⁵. Moreover, the comparison of multiple samples is not easy.

The super-SILAC approach emerged as a variant applicable to tissue samples or biological fluids ²⁴⁶. This approach consists in combining an assortment of cell lines, a super-SILAC mix, to be used as a spike-in standard. The design of the appropriate super-SILAC mix is crucial for the outcome of the experiment. The cell line mixture that better represents the

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proteome of the clinical sample, the ratios in which these cells should be mixed, and the ratio of the super-SILAC to be spiked in the clinical sample must be determined ²⁴⁷. It is an appropriate approach for untargeted studies such as discovery phases of the biomarker pipeline (Figure 13B), since this approach presents the advantages of conventional SILAC, enabling the relative quantification of the complete proteome. Moreover, this approach allows for the comparison of multiple samples. Its main disadvantage is that the dilution of the sample of interest by a complete exogenous proteome significantly increases the complexity of the sample, and thus may reduce the selectivity and the sensitivity of the approach.

! Isotope-labeled synthetic peptides

Isotope-labeled peptides can be chemically synthesized in large scale by several manufacturers on the market with different quality grades ranging from relatively inexpensive non-purified peptides used for relative quantification (crude peptides) to purified and accurately quantified peptides designed for absolute quantification (e.g., AQUA peptides)

248,249. Furthermore, post-translational modifications such as phosphorylation, acetylation, sulfation can be incorporated ²⁵⁰. It is a fast and straightforward approach that introduces less complexity to the sample compared to the super-SILAC approach, and it is widely used nowadays in targeted studies where the peptides of interest are known upfront, such as the verification and validation of biomarker candidates (Figure 13B). However, this approach also has some limitations. Synthetic peptides as internal standards do not control for variations in early steps of the sample preparation, including the tryptic digestion.

Additionally, synthesis of peptides over 25 amino acids can be erratic, the stability during storage should be controlled, and the preparation of a mixture of hundreds of peptides can be a tedious task if not automated.

! Isotopically labeled derivatization reagents

The isobaric tags for relative and absolute quantitation (iTRAQ) technology utilizes isobaric reagents to label the primary amines of peptides and proteins ²⁵¹. The isobaric tags include a reporter group, a mass balance group and a peptide-reactive group. The function of the balance group is to make the labeled peptides from each sample isobaric (same mass). The relative abundance of the peptides is deduced from the relative intensities of the reporter group that is generated upon fragmentation in the mass spectrometer. The iTRAQ approach is often used in discovery studies since, in principle, every peptide is labeled. However, it also has some limitations, such as the underestimation of the changes in abundance reported and interferences by cross-label isotopic impurities ²⁵². Other approaches based on

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isotopically labeled reagents include isotope coded affinity tagging (iCAT) ²⁵³ and tandem mass tags (TMT) ²⁵⁴.

5.1.2.2. Label-free approaches

In label-free approaches, an equal amount of each sample is analyzed by MS to estimate the relative abundance of proteins across samples, without the use of a labeling strategy ²⁵⁵. The sample processing and LC-MS conditions need to be carefully controlled to minimize variations and obtain optimal results ²⁵⁶.

Although this method provides a less accurate quantification, its ease of execution and cost-effectiveness make it appropriate for the initial discovery phase of the biomarker pipeline (Figure 13B). The label free approach is sensitive to MS signal variation and therefore, it is more reliable when samples with similar chemical background are analyzed. Due to its limited precision and accuracy, the difference between the levels of the proteins among samples must be high to be significant (greater than two-fold) ²⁵⁷.

5.1.3. Major mass spectrometry acquisition strategies in