Key words

1 Introduction

Since the mid-1990s, proteomics by mass spectrometry has been an extremely useful tool in the discovery of new biomarkers and for elucidating new disease mechanisms. Many papers have been published using untargeted proteomic (more recently referred to as hypothesis generating) MS workflows which can demonstrate and detect differences between disease and control cases in neurodegenerative disorders [1,2,3]. Until recently validation of possible biomarkers has mainly been possible by immunological means, such as western blot or enzyme-linked immunosorbent assay (ELISA). However, mass spectrometry is proving to be ever more useful in providing accurate and specific quantitative data on target proteins of interest in body fluids [4]. In particular this has proved of great interest to those researchers studying the composition of cerebrospinal fluid (CSF) to find biomarkers for Alzheimer’s disease (AD) [3] and in the development of new tests for this disease.

Targeted proteomics (single reaction monitoring (SRM) detecting one analyte and multiple reaction monitoring (MRM), detecting more than one analyte) is a tandem mass spectrometry (MS/MS) technique usually performed on a triple quadrupole MS-based instrument. Tandem mass spectrometry has been used for more than 20 years [5, 6] but almost exclusively for the quantitation of small molecules, such as metabolites and drugs. However it is only recently that tandem mass spectrometry has been used more and more to quantify proteolytically generated peptides that act as surrogates of the corresponding intact proteins. The use of stable isotope-labelled peptide standards can make this method of quantitation not only extremely accurate but also more specific than conventional immunological-based assays.

This technique is generally used in conjunction with the hypothesis generating or proteomics-based technology such as label-free liquid chromatography-tandem mass spectrometry (LC-MS/MS) or label-based proteomic methods such as stable isotope labelling with amino acids in cell culture (SILAC) and tandem mass tagging (TMT). These types of methods provide protein identification and relative protein abundance using the spectral count, stable isotope labelling, or the total number of MS/MS spectra taken on peptides from a given protein in a given LC-MS/MS analysis. This is then linearly correlated with protein abundance but does not give absolute quantification [7,8,9]. For the development of targeted proteomics or peptide-based MS quantitation, the first step is proteolytic digestion of the proteins by a protease, typically trypsin, which allows the creation of fragments small enough to be analyzed within the mass range of triple quadrupole-based instruments. Trypsin cleaves at the carboxylic acid side of peptide bonds of lysine and arginine residues and is generally used due to its robustness, specificity, and the creation of peptides that are amenable to mass spectrometry. The peptides produced are now of optimal size and molecular weight and in the range of most quadrupole or ion trap based tandem mass spectrometers. Once the protein has been digested, it is possible to choose a “marker” peptide for that protein. This marker can then be weighed in the first mass analyzer (MS1) and its unique mass-to-charge (m/z) ratio recorded. The peptide is then fragmented in a collision cell by colliding it with argon gas to yield product ions which have their m/z ratio recorded in the second part of the mass spectrometer or MS2. This fragmentation is known as collision-induced dissociation (CID), and from this, a unique product ion is chosen as being the characteristic fingerprint of that peptide. Here we have two levels of identification for the peptide, firstly its intact mass—precursor ion—and secondly it is identifying peptide fragment mass, product ion. In method development more than one precursor and product ion will be chosen to optimize, and the most specific/sensitive will be chosen. Finally, a third and final level of identification is given by the elution time of the peptide from the UPLC chromatography column. Because in a complex biological sample digest, it is possible to get several peptides with the same precursor/product masses as your target, this third identifying step is very important. It is the use of fast chromatography-based systems such as UPLC with tandem mass spectrometry that provides extra specificity for the analysis of proteins that immunological-based assays cannot match. However, it should be noted that some immunological-based assays can be significantly more sensitive than MS-based analyses.

TREM2 is a receptor glycoprotein of 230 amino acids which belongs to the immunoglobulin superfamily. In the brain, TREM2 is expressed exclusively by myeloid cells which include microglia the brains resident immune cell [10]. It was known that homozygous mutations in this protein caused polycystic lipomembranous osteodysplasia with sclerosing leukoencephalopathy, also known as Nasu-Hakola disease [11, 12]. However, in 2013, it was discovered that heterozygous mutations in the TREM2 gene were noted as being significant risk factors for AD [13, 14].

This discovery led to intense interest in TREM2 and a requirement for sensitive assays to measure the soluble protein. In 2013 there were very few immunoassays available, and those that were available were not robust. Therefore, a different method of measuring sTREM2 was required, and this led to the development of an SRM technique that was published in early 2016 [15]. The benefit of this method is the huge scope for multiplexing as shown by Heywood et al. in [3]. Although this chapter is using sTREM2 as an example for the method, other interesting proteins can easily be multiplexed and augmented this way.

2 Materials

2.1 Mass Spectrometer

Targeted proteomics were performed on a Waters ultraperformance liquid chromatography system (Manchester, UK) coupled to a Waters XEVO TQ-S triple quadrupole mass spectrometer. The column used was a Waters CORTECS UPLC C18+ column, 90 Å 3 mm × 100 mm attached to a C18+ VanGuard pre-column.

2.2 Samples

CSF samples were collected from memory clinics in Sweden and the UK according to local ethics procedures. Samples were collected in the morning into polypropylene tubes (Sarstedt, Numbrecht, Germany) according to standard operating procedures; samples were then further aliquoted into polypropylene tubes, frozen, and stored at −80 °C within 2 h.

2.3 Reagents

All reagents are of mass spectrometry grade and prepared and stored at room temperature unless otherwise stated.

2.3.1 Digestion Reagents

Digestion buffer—100 mM Tris pH 7.8 containing 6 M urea, 2 M thiourea, and 2% amidosulfobetaine-14 (ASB-14). 2 M dithiothreitol (DTT) is made by dissolving 30 mg of DTT in 1 mL of 100 mM Tris pH 7.8. It is important that this solution is made up fresh on the day and kept on ice until use. Iodoacetamide (IAA) was prepared by dissolving 36 mg in 1 mL of 100 mM Tris pH 7.8; similarly this solution was made up fresh on the day and protected from light. Sequence grade trypsin (Promega, UK) is prepared by resuspending lyophilized 1 mg aliquots of the protease with 11 μL of 40 mM ammonium bicarbonate buffer. All reagents are from Sigma-Aldrich, Dorset, UK, unless otherwise stated.

2.3.2 Mass Spectrometry Reagents

MS mobile phases are A, LC-MS grade water with 0.1% formic acid (FA), and B, LC-MS grade ACN with 0.1% FA.

2.3.3 Peptides and Proteins

TREM2 peptide VLVEVLADPLDHR was synthesized by Generon (Maidenhead, UK). Yeast enolase standard was from Sigma-Aldrich (Dorset, UK).

3 Methods

Figure 1 shows a typical SRM development workflow, while Fig. 2 depicts an assay workflow.

Fig. 1
figure 1

Typical single reaction monitoring (SRM) development workflow

Full size image
Fig. 2
figure 2

Typical assay workflow

Full size image

3.1 SRM Set Up

To set up the SRM assay for sTREM2, we first identified a unique peptide from the protein sequence using Skyline, https://skyline.ms/project/home/software/Skyline/. Skyline is a freely available and open source Windows client application for building selected reaction monitoring (SRM)/multiple reaction monitoring (MRM) and parallel reaction monitoring (PRM—targeted MS/MS), although, it would be preferable to identify peptides from previous mass spec discovery experiments. Another useful resource is the Global Proteome Machine http://www.thegpm.org/. We checked that the peptide was completely unique to TREM2 and that none of the amino acids would be subject to posttranslational modification. This peptide was synthesized by Generon (Berkshire, UK) and then used to develop the assay.

For tuning of the peptide, standards are reconstituted in 50% ACN containing 0.1% FA before being infused into the mass spectrometer. The amount of peptide will need to be optimized but a suggested starting point is 100 μg/mL. A full scan can be performed initially to optimize tuning concentration, and the peptide is diluted accordingly. Once the concentration has been optimized, optimal collision and cone energies are obtained using the automatic “Intellistart” tuning software of the Xevo TQS mass spectrometry system. After the optimal fragmentation and MS parameters are obtained and entered into the SRM, the retention time of the peptide is assessed. In this case a 10 min chromatographic run time is used, which is shown in Table 1 (Notes 1 and 2 ).

Table 1 10-minute chromatographic run
Full size table

The advantage of using peptides generated from actual digests, under typical laboratory conditions, is that it allows the user to identify the peptides that are amenable to mass spectrometry, i.e., in silico determined tryptic fragments cannot predict chromatograph peak shape or fragmentation patterns. This often saves considerable time in method development (Note 3 ).

Once the assay has been developed using synthetic peptides, it is important to then ensure your peptide can be detected in the appropriate sample matrix. In this case, the synthetic peptide is used to test this by spiking varying amounts in to CSF. It is also necessary to optimize the amount of your sample to dry down and the amount of digest to inject onto the column (Notes 4 – 6 ).

3.2 Sample Preparation

100 μL of each CSF sample is aliquoted into a fresh siliconized Eppendorf tube and freeze dried. The resulting proteins are then resuspended in 20 μL of digestion buffer and shaken at RT for 20 min. 1.5 μL of 2 M DTT is added to reduce disulfide bridges and the sample shaken for a further hour at RT. 3 μL of (2 M) IAA is added in order to carboamidomethylate any cysteines contained in the proteins, and the sample is then incubated at RT for a further 45 min in the dark. The sample is then diluted with 165.5 μL of LC-MS grade water and 10 μL of a solution containing 1 μg of trypsin. Samples are then vortexed and left in a water bath at 37 °C overnight. Before mass spectral analysis, centrifuge samples at 13,000 × g for 10 min to remove particulates before transfer into vials suitable for mass spectral analysis.

3.3 Mass Spectrometry

Using the chromatographic and mass spectral conditions developed and described above, the samples can be input into the sample manager and the analyses completed. During any analyses a standard curve of your peptide should also be included, and this should cover the range that the endogenous levels of peptides are expected to be present in your subject samples. Each standard curve is made up in pooled CSF in order that any matrix ion suppression is accounted for. It is also good practice to create quality controls as well using pooled CSF spiked with your peptide(s) in order that you can check the sensitivity of the assay at appropriate intervals (should fall within ±10% of established quantities). It is recommended to run QC samples at intervals of 10–15 subject samples. Internal standard values should also be monitored, and coefficients of variation (%CV) of these should also be used to monitor assay stability.

3.4 Data Analysis

Data analysis uses Waters MassLynx and TargetLynx V4.1 software to process the raw data. Methods can be set up so that the integrated peaks can be ratioed to the internal standard which can be yeast enolase in this case or a stable isotope-labelled peptide. Integration should be checked to confirm accuracy, and then response values can be interpolated from your standard curve.

4 Conclusion

In summary, the SRM method for the quantification of sTREM2 described in this chapter has been used to investigate levels of sTREM2 in a cohort of dementia subjects and controls and later validated on a separate cohort. More recently this protein has been found to be increased in early AD [16, 17] and highlights the importance of inflammation as an early event in this disease. This is a highly adaptable method, and multiplexing is a matter of adding suitably optimized MRM files as shown above.

5 Notes and Troubleshooting

  1. 1.

    For one transition the method time can be shortened, say 5 min, when multiplexing; it is suggested that MRM files should be grouped and split according to retention time, in order to maximize the dwell time of each ion.

  2. 2.

    Transitions should be selected on the best intensity and absence of interfering peaks.

  3. 3.

    Synthetic peptides that contain cysteine residues must be carboamidomethylated prior to infusion.

  4. 4.

    If matrix interference makes analyte detection a problem, then the MS resolution can be reduced; this results in an increase of sensitivity. Accurate mass will need to be determined for the synthetic peptide at the new resolution.

  5. 5.

    The least amount of sample to enable the analyte to be detected should be injected onto the column. Too much sample can lead to ion suppression effects. In a multiplex situation, it is worth grouping transactions according to the endogenous abundance of the analyte in the sample, in order that you can optimize the amount of sample to inject.