This research was supported by the Intramural Research Program of the NIH, National Institute of Allergy and Infectious Diseases.

NOTE: This method has been previously described56.
1. Peptide Target Selection
<ol>
	<li>Compile a list of the target proteins, and include a small number of housekeeping proteins for normalization across biological samples, and also include an internal protein standard (e.g., firefly luciferase). Digest the target proteins into tryptic peptides in silico using a software tool such as Protein Digestion Simulator29,30.</li>
	<li>Require that the peptide...

Absolute protein quantification is essential for a very diverse range of biomedical applications such as biomarker validation and signal transduction pathway modeling. Recently, targeted proteomics using LC-SRM has benefited from improvements to numerous technologies including peptide standard preparation, HPLC, QqQ-MS, and LC-SRM data analysis. Consequently, it has become a powerful alternative to immunoassays. Immunoassays can be extremely sensitive and high-throughput, but developing a robust immunoassay can be extrem...

Proteomic experiments that use mass spectrometry (MS) can be designed to use either non-targeted (shotgun) or targeted methods. Discovery proteomics generally relies on bottom-up shotgun MS, either by using a traditional data-dependent acquisition mode, or by using one of the recently developed data-independent techniques (e.g., MSE, SWATH)1,2. Shotgun proteomics is a powerful tool for high-throughput peptide identification and relative quantification, but it is generally unsuitable for absolute quantification or for targeting small, defined sets (~tens) of proteins. The MS method most often used for targeted proteomics is selected reaction monitoring (SRM) because of its high sensitivity, speed, and dynamic range3-5. Alternatives to SRM include parallel reaction monitoring, which takes advantage of high-resolution, full MS scanning6.
SRM is usually performed using a nano-flow reversed-phase high-performance liquid chromatography (nano-RP-LC) instrument coupled to a nano-electrospray ionization (nano-ESI) ion source attached to a triple quadrupole mass spectrometer (QqQ-MS). In a typical experiment, sample proteins are proteolytically digested, and the resulting peptides are chromatographically separated, desorbed, and ionized. The resulting precursor ions are m/z filtered by the first quadrupole (Q1) and fragmented in the second quadrupole (q2) by colliding them with a collision gas. The resulting fragment ions are m/z-filtered in the third quadrupole (Q3) and quantified by a dynode. Each precursor and fragment ion pair is referred to as a transition, and each transition is monitored for a specified time period (the dwell time; typically 2-50 msec). During LC-SRM, the QqQ-MS cycles through a predefined list of transitions (the duty cycle is typically &#8804;3 sec), and a chromatogram of each transition is produced.
Alternative strategies for protein quantification typically use immunoassays such as dot blots, Western blots, ELISAs, antibody microarrays, reverse phase protein microarrays, microfluidic immunoassays, digital ELISAs, and microsphere-based immunoassays7. The best immunoassays can be significantly more sensitive than LC-SRM, and sample throughput of immunoassays can be significantly higher than that of LC-SRM5. However, developing immunoassays can be expensive and/or time consuming, and the resulting assays can be vulnerable to cross-reactivity and/or interference, incompatible with cell/tissue lysis/homogenization methods, and/or not amenable to multiplexing5,8. Some of these issues can be addressed by coupling antibody- and MS-based techniques. For example, target proteins can be enriched using immunoprecipitation prior to proteolysis and LC-SRM9-12. Alternatively, the SISCAPA technique employs immunoprecipitation subsequent to proteolysis at the peptide level13,14. In addition to immunoenrichment strategies, immunodepletion of high abundance proteins can be employed to increase LC-SRM sensitivity by reducing interference by coeluting analytes15,16.
MS-based protein quantification can be divided into relative and absolute quantification, and also into label-free and stable isotope labeling (e.g., metabolic labeling, chemical labeling, and heavy-labeled protein and peptide internal standards). Label-free techniques can be useful for relative protein quantification, but are unsuitable for accurate absolute quantification. By comparison, labeling techniques have reduced error associated with sample preparation and MS variance, and are often used for relative protein quantification17. For example, stable isotope labeled proteome (SILAP) standards prepared using a cultured human cell line enabled relative quantification of potential biomarkers via LC-SRM of human serum18. Accurate absolute protein quantification by MS requires that purified, quantified, isotopically-labeled protein or peptide internal standards be spiked-into biological samples prior to MS. The incorporation of heavy isotope labeled internal standards into an LC-SRM workflow enables absolute quantification that has been shown to be highly reproducible and transferable between laboratories16,19.
Stable isotope labeled internal standards for absolute protein quantification by MS include peptide standards prepared using solid phase synthesis20, proteins composed of concatenated protease-cleavable peptide standards21, and full-length protein standards22. Target protein covalent modification and incomplete sample preparation (i.e., incomplete sample lysis and homogenization, and incomplete protein solubilization, denaturation, alkylation, and proteolysis) can undermine accurate quantification. Internal protein standards are the least likely to be affected by most of these potential problems, but they are usually the most difficult to prepare. An alternative is to analyze each target protein using multiple internal peptide standards which are designed to include amino- and carboxy-terminal native flanking residues. Regardless of which type of internal standard is employed, it should be spiked-into the biological samples at as early a point during sample preparation as possible. Also, multiple sample preparation techniques (e.g., different denaturation conditions) should be tested. The usage of multiple orthogonal experimental techniques (experimental cross-validation) is a viable strategy for overcoming most potential quantification challenges23-25.
LC-SRM quantification of proteins is a highly flexible technique that has been used in a wide variety of applications. Notably, it has been used to study peptide and protein biomarkers within clinical samples such as serum, core biopsies, and fine needle aspirates5. LC-SRM has also been used to measure the stoichiometry of protein complexes5,26, to detect botulinum neurotoxins27, to quantify protein phosphorylation dynamics within signaling pathways5, and to quantify changes in protein conformation28.
Our laboratory is using LC-SRM to quantify the signaling proteins that mediate macrophage chemotaxis to support the development of chemotaxis pathway simulations. The overall scheme of the protocol (Figure 1) begins with ranking the tentative target peptides. Subsequently, crude external peptide standards are synthesized and used to develop LC-SRM assays for qualitative analyses of biological samples. If the biological sample-derived target peptide is detected, purified heavy-labeled internal peptide standards are prepared for quantitative LC-SRM. This protocol can be used to accurately quantify proteins from a wide variety of biological samples, and to support investigations of a wide variety of protein targets.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Acetonitrile (ACN), LC-MS grade</td>
			<td>Fisher</td>
			<td>A955-1</td>
			<td></td>
		</tr>
		<tr>
			<td>BCA (bicinchoninic acid) protein assay kit</td>
			<td>Fisher</td>
			<td>23235</td>
			<td></td>
		</tr>
		<tr>
			<td>Beads for bead beating, zirconia-silica, 0.1mm</td>
			<td>BioSpec Products</td>
			<td>11079101z</td>
			<td></td>
		</tr>
		<tr>
			<td>Bestatin hydrochloride</td>
			<td>Sigma</td>
			<td>B8385-10MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture DMEM (with glucose, without L-glutamine)</td>
			<td>Lonza</td>
			<td>12-614F</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture EDTA, 500mM, pH8</td>
			<td>Gibco</td>
			<td>15575</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture fetal bovine serum (FBS)</td>
			<td>Atlanta Biologicals</td>
			<td>S11550</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture L-glutamine</td>
			<td>Sigma</td>
			<td>G8540-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture phosphate buffered saline (PBS) pH 7.4</td>
			<td>Gibco</td>
			<td>10010-049</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture Trypan Blue viability stain, 0.4% w/v</td>
			<td>Lonza</td>
			<td>17-942E</td>
			<td></td>
		</tr>
		<tr>
			<td>Cellometer Auto T4 cell counter</td>
			<td>Nexcelom Bioscience</td>
			<td>Cellometer Auto T4</td>
			<td></td>
		</tr>
		<tr>
			<td>Cellometer Auto T4 disposable counting chambers</td>
			<td>Nexcelom Bioscience</td>
			<td>CHT4-SD100-014</td>
			<td></td>
		</tr>
		<tr>
			<td>Dithiothreitol (DTT)</td>
			<td>Sigma</td>
			<td>D5545-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>Formic acid, LC-MS grade, ampules</td>
			<td>Fisher</td>
			<td>A117-10X1AMP</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemocytometer, Neubauer-improved, 0.1mm deep</td>
			<td>Marienfeld-Superior</td>
			<td>0640030</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES, 1M, pH 7.2</td>
			<td>Mediatech</td>
			<td>25-060-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric acid, 37% w/w</td>
			<td>VWR</td>
			<td>BDH3028-2.5LG</td>
			<td></td>
		</tr>
		<tr>
			<td>Iodoacetamide</td>
			<td>Sigma</td>
			<td>I1149-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>Laser Based Micropipette Puller</td>
			<td>Sutter Instrument Co.</td>
			<td>P-2000</td>
			<td></td>
		</tr>
		<tr>
			<td>LC Magic C18AQ, 5&#181;m, 200&#8491;, loose media</td>
			<td>Michrom Bioresources</td>
			<td>PM5/61200/00</td>
			<td></td>
		</tr>
		<tr>
			<td>LC Halo ES-C18, 2.7&#181;m, 160&#8491;, loose media</td>
			<td>Michrom Bioresources</td>
			<td>PM3/93100/00</td>
			<td></td>
		</tr>
		<tr>
			<td>LC coated silica capillary, 50&#181;m id</td>
			<td>Polymicro Technologies</td>
			<td>1068150017</td>
			<td></td>
		</tr>
		<tr>
			<td>LC vial, autosampler, 12x32mm polypropylene</td>
			<td>SUN SRI</td>
			<td>200-268</td>
			<td></td>
		</tr>
		<tr>
			<td>LC vial screw cap, autosampler, pre-slit PTFE/silicone</td>
			<td>SUN SRI</td>
			<td>500-061</td>
			<td></td>
		</tr>
		<tr>
			<td>Luciferase, from Photinus pyralis</td>
			<td>Sigma</td>
			<td>L9506-1MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Pepstatin A</td>
			<td>EMD Millipore</td>
			<td>516481-25MG</td>
			<td></td>
		</tr>
		<tr>
			<td>pH strips colorpHast (pH 0.0-6.0)</td>
			<td>EMD Chemicals</td>
			<td>9586-1</td>
			<td></td>
		</tr>
		<tr>
			<td>PhosStop phosphatase inhibitor cocktail</td>
			<td>Roche</td>
			<td>04906837001</td>
			<td></td>
		</tr>
		<tr>
			<td>RapiGest SF</td>
			<td>Waters</td>
			<td>186001861</td>
			<td></td>
		</tr>
		<tr>
			<td>Sep-Pak SPE, C18 1ml 100mg cartridge</td>
			<td>Waters</td>
			<td>WAT023590</td>
			<td></td>
		</tr>
		<tr>
			<td>Sep-Pak SPE, extraction manifold, 20 position</td>
			<td>Waters</td>
			<td>WAT200609</td>
			<td></td>
		</tr>
		<tr>
			<td>Sep-Pak SPE, flat-surfaced rubber bulb</td>
			<td>Fisher</td>
			<td>03-448-25</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydroxide (NaOH)</td>
			<td>Fisher</td>
			<td>S318-500</td>
			<td></td>
		</tr>
		<tr>
			<td>SpeedVac vacuum concentrator</td>
			<td>Fisher</td>
			<td>SPD111V</td>
			<td></td>
		</tr>
		<tr>
			<td>Trifluoroacetic acid (TFA), LC-MS grade</td>
			<td>Fisher</td>
			<td>A116-50</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin, sequencing grade, modified</td>
			<td>Promega</td>
			<td>V5113</td>
			<td></td>
		</tr>
		<tr>
			<td>Tube decapper for Micronic tubes</td>
			<td>USA Scientific</td>
			<td>1765-4000</td>
			<td></td>
		</tr>
		<tr>
			<td>Tubes, 2ml microcentrifuge, o-ring screw-cap, sterile</td>
			<td>Sarstedt</td>
			<td>72.694.006</td>
			<td></td>
		</tr>
		<tr>
			<td>Urea</td>
			<td>Sigma</td>
			<td>U0631-500g</td>
			<td></td>
		</tr>
		<tr>
			<td>Water, LC-MS grade</td>
			<td>Fisher</td>
			<td>W6-1</td>
			<td></td>
		</tr>
	</tbody>
</table>

selected reaction monitoring mass spectrometry for absolute protein quantification

Absolute quantification of target proteins within complex biological samples is critical to a wide range of research and clinical applications. This protocol provides step-by-step instructions for the development and application of quantitative assays using selected reaction monitoring (SRM) mass spectrometry (MS). First, likely quantotypic target peptides are identified based on numerous criteria. This includes identifying proteotypic peptides, avoiding sites of posttranslational modification, and analyzing the uniqueness of the target peptide to the target protein. Next, crude external peptide standards are synthesized and used to develop SRM assays, and the resulting assays are used to perform qualitative analyses of the biological samples. Finally, purified, quantified, heavy isotope labeled internal peptide standards are prepared and used to perform isotope dilution series SRM assays. Analysis of all of the resulting MS data is presented. This protocol was used to accurately assay the absolute abundance of proteins of the chemotaxis signaling pathway within RAW 264.7 cells (a mouse monocyte/macrophage cell line). The quantification of Gi2 (a heterotrimeric G-protein &#945;-subunit) is described in detail.

The development of predictive computational models of signal transduction pathways is one of the fundamental goals of systems biology53. Unfortunately, even for signaling pathways that have been studied extensively and have a high clinical significance, it is still not generally possible to quantitatively predict pathway behavior in response to perturbations (e.g., this is true for the MAPK/ERK pathway54). Recently, an investigation employed targeted proteomics, transcriptomics, and computa...

Watch this Scientific Journal Video about Selected Reaction Monitoring Mass Spectrometry for Absolute Protein Quantification at JoVE.com

Selected Reaction Monitoring Mass Spectrometry for Absolute Protein Quantification

This protocol describes how to perform absolute quantification assays of target proteins within complex biological samples using selected reaction monitoring. It was used to accurately quantify proteins of the mouse macrophage chemotaxis signaling pathway. Target peptide selection, assay development, and qualitative and quantitative assays are described in detail.

Absolute quantification of target proteins within complex biological samples is critical to a wide range of research and clinical applications. This ...

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16.9K visualizações.  National Institutes of Health. This protocol describes how to perform absolute quantification assays of target proteins within complex biological samples using selected reaction monitoring. It was used to accurately quantify proteins of the mouse macrophage chemotaxis signaling pathway. Target peptide selection, assay development, and qualitative and quantitative assays are described in detail.