A Versatile Mass Spectrometry Sample Preparation Procedure for Complex Protein Samples

Prepare high-quality peptide digests with high recovery from cultured cells.

by Babu Antharavally, Ph.D.; Xiaoyue Jiang, Ph.D.1; Robert Cunningham, Ph.D.; Ryan Bomgarden, Ph.D.; Yi Zhang, Ph.D.1; Rosa Viner, Ph.D.1; John C. Rogers, Ph.D. - 06/04/13

Mass spectrometry (MS) has become a prominent technique in biological research for the identification, characterization, and quantification of proteins (Ref. 1). Shotgun proteomics is a commonly used strategy to identify proteins in complex mixtures by digesting proteins at specific amino acids into peptides that can be separated and identified by mass spectrometry (Ref.2). In order to identify thousands of proteins from a complex lysate, it is essential to have robust sample preparation methods for protein extraction, reduction, alkylation, digestion, and clean-up. The quality and consistency of sample preparation influences the time and cost of MS analysis and the reliability and accuracy of results. For MS-based proteomics to reach its full potential as a routinely used detection technology in research and clinical settings, the variability associated with the sample preparation steps that precede MS analysis must be addressed. Despite extensive literature describing various MS sample preparation methods, there is little standardization among methods, resulting in confusion for those who are new to MS sample preparation techniques, and high variability in MS analysis results, even among expert MS sample prep labs.

Proper sample preparation includes efficient extraction of proteins, complete reduction of disulfide bonds, selective alkylation of cysteines without non-specific modification of other amino acids, reproducible proteolysis, and complete removal of contaminants including detergents, lipids, and salts prior to MS analysis.

Here we describe a simple, versatile, and robust protocol to produce clean, reproducible peptide mixtures for MS (Figure 1), which we have commercialized as the Thermo Scientific Pierce Mass Spec Sample Prep Kit for Cultured Cells (Part No. 84840). This Pierce procedure incorporates two-stage enzymatic digestion with LysC and trypsin proteases. The method also involves using an internal control-protein, called a Digestion Indicator (Part No. 84841), to monitor and compare the efficiency of sample prep experiments.

Figure 1. Diagram of the developed protocol. Reagents and instructions for this procedure have been commercialized as the Thermo Scientific Pierce Mass Spec Sample Prep Kit for Cultured Cells (Part No. 84840).

We compared performance of the Pierce protocol to three other popular MS sample preparation methods: filter-assisted sample preparation (FASP)(Ref.3), ammonium bicarbonate (AmBic)/SDS (Ref.4), and urea extraction (Table 1). The optimized Pierce protocol is highly consistent, scalable, compatible with downstream processing, and versatile enough to process tissue samples.

There are many examples of proteomic sample preparation methods that have been described in the literature (Refs. 2-4), and it is not uncommon for these methods to be modified by subsequent members of the same lab or by other laboratories. This makes it extremely difficult for new MS users to find the best protocol and use it to obtain consistent results. Each of these common protocols has disadvantages; FASP requires many long centrifugation steps, SDS-based methods may not be scalable and require detergent removal from peptides, and urea must be made fresh and can carbamylate lysine residues. Because sample preparation is the most problematic area of MS-based proteome analysis, it is important to have robust, reproducible methods that can be easily adopted by novice and expert MS labs alike.

Summary of the optimized Pierce Kit sample preparation protocol compared to three other popular proteomic sample prep methods that were evaluated in this study.

To develop the Pierce protocol, we first used a step-wise approach to optimize a cell lysis method to maximize protein extraction and recovery from the resulting lysate. This protocol, based on a proprietary Lysis Buffer plus heat and sonication (Figure 1) can extract significantly more cellular protein than FASP, AmBic/SDS and urea methods (Figure 2). Results from Jurkat and NIH 3T3 cells were comparable to HeLa cells (data not shown).

Next we assessed the completeness of disulfide reduction, the selectivity of alkylation at cysteine residues, and the digestion efficiency with single (trypsin) and double digestion (LysC-trypsin) routines. In initial studies we optimized the conditions for trypsin digestion of cellular lysate proteins with LC-MS/MS analysis on Thermo Scientific Velos Pro and Thermo Scientific Orbitrap XL instruments. This analysis indicated <10% missed cleavages. However, we observed 20-25% missed cleavages when the same samples were analyzed on Thermo Scientific Q Exactive or Orbitrap Elite instruments. Numerous experiments consistently found that these faster, high-resolution instruments identify many long, higher charged peptides with missed cleavages that are not detected on lower-resolution, slower mass spectrometers. Therefore, we developed and optimized the double-digestion LysC-trypsin protocol until it consistently resulted in less than 10% missed cleavages on Thermo Scientific Q Exactive and Orbitrap Elite instruments. We carefully optimized the reaction buffers and protocol for high-resolution spectrometers to minimize non-selective alkylation or incomplete digestion. The final reagent formulations and overall protocol significantly improved the reproducibility and number of peptide and protein identifications compared to the existing methods (Tables 2 and 3).

Table 2. Comparison of peptide and protein identification results by four MS sample prep methods.

From one source culture of HeLa cells, triplicate pellets (2 x 10^6 cells each) were lysed by each method. Subsequently, 100µg amounts of each replicate lysate were processed by the respective protocol. Finally, 500ng samples were analyzed by LC-FT MS/IT MS2 CID on a Thermo Scientific Orbitrap Elite mass spectrometer.

Table 3. Reproducibility of LC-MS/MS results from three biological replicates.

From one source culture of HeLa cells, triplicate pellets (2 x 10^6 cells each) were lysed by the Pierce protocol. Resulting lysate samples (200µg in 200µL of Lysis Buffer) were spiked with 2μg Digestion Indicator and processed through remaining steps of the Pierce protocol. Finally, 500ng samples were analyzed by LC-MS/MS on a Thermo Scientific Q Exactive mass spectrometer.

To aid in testing and comparison of protocol conditions and experimental runs, we developed a Digestion Indicator (Part No. 84841), which is included as part of the kit. This indicator is a non-mammalian protein that can be spiked into lysates (see Figure 1) and carried through the sample prep procedure, which results in five (5) distinct peptides that can be quantified.

For example, to test reproducibility of our optimized method, we processed and analyzed quadruplicate samples of a HeLa cell culture using the Pierce protocol, spiking the Digestion Indicator into each lysate after the initial lysis step (same method as for Table 3). We then analyzed these samples by LC-MS/MS on a Thermo Scientific Velos Pro ion trap mass spectrometer. Digestion indicator peptides were quantified with Thermo Scientific Pinpoint 1.2 software, which is pre-programmed with information on the Digestion Indicator peptides and MS2 transitions to quantify (Figure 3). The coefficients of variation (CV) for replicates of the five peptides were 5-16% with an overall mean CV of 10% (Table 4). This quantitative analysis further demonstrated the high reproducibility of sample processing using the optimized protocol.

Table 4. Digestion Indicator peptides and example assessment of reproducibility.

Sequences of the five peptides that result from the Digestion Indicator, and coefficients of variation (CV) for triplicate samples processed using the Pierce protocol (Part No. 84840).

To assess the scalability of the reduction, alkylation, digestion steps in the sample preparation protocol, we processed five different amounts of HeLa cell lysate (10, 50, 100, 200 and 5000µg) using the method. Analysis of equivalent volumes of peptide samples by LC-MS/MS resulted in identical chromatograms, demonstrating the scalability of this protocol over a 500-fold dynamic range (Figure 4).

Figure 4. Scalability of MS sample prep kit protocol. Hela lysate samples (10µg-5mg) were prepared according to the Pierce protocol (Part No. 84840). Samples (500ng) were subjected to LC-MS/MS analysis on a Thermo Scientific Velos Pro ion trap mass spectrometer.

Finally, we tested the protocol with brain tissue, which resulted in reproducible, high quality peptide sample preparations, demonstrating the versatility of this method for different cell and tissue sample types (Figure 5).

Figure 5. Evaluation of sample preparation workflow with tissue samples. Two samples of mouse brain tissue (0.25g) were homogenized with a tissue tearer and the proteins were extracted using the Thermo Scientific Pierce Mass Spec Sample Prep Kit for Cultured Cells (Part No. 84840). Then, 100µg of lysate was processed according to the kit procedure, and 500ng samples were analyzed by LC-MS/MS on a Thermo Scientific Velos Pro ion trap mass spectrometer.

We have developed an optimized protocol and kit of reagents that standardize peptide sample preparation for MS analysis (Figure 1). This protocol reproducibly yields high-quality peptide samples for LC-MS/MS analysis that provide high rates of protein identification as a result of efficient and selective protein extraction, reduction, alkylation, and digestion (Table 3). This method yields more protein lysate from cultured cells, is highly reproducible, is scalable from 10µg to 5mg, is simpler and faster than FASP, has no risk of carbamylation by urea, and results in higher protein identification rates than other popular “standard” sample preparation methods (Figure 2 and Table 2). This protocol also includes a unique, non-mammalian internal digestion control standard protein (Digestion Indicator) to assure protocol performance and to quantify sample preparation processing and digestion efficiency across samples.

The complete Pierce Mass Spec Sample Prep Kit for Cultured Cells includes Lysis Buffer, Digestion Indicator, Reaction Buffers, Proteases and with instructions to process up to 20 samples. The simple protocol is user-friendly for non-expert MS analysts, making this ideal for proteomics core lab clients. The final prepared samples are ready for direct MS analysis or other downstream applications, including peptide fractionation, mass-tag labeling, or phosphopeptide enrichment.

Protein extraction

Duplicate or triplicate HeLa S3 cell pellets, each containing  2 x 106 cells, were suspended in respective method lysis buffers:

• FASP: 0.2mL of 0.1M Tris-HCl, 4% SDS, 0.1M DTT, pH 7.6
• AmBic-SDS: 0.05M ammonium bicarbonate, 0.1% SDS, pH 8.0
• Urea: 0.1M Tris-HCl, 8M urea, pH 8.5
• Pierce: Lysis Buffer from the Thermo Scientific Pierce Mass Spec Sample Prep Kit for Cultured Cells (Part No. 84840)

Samples were incubated at 95°C for 5 minutes except the urea sample, which was incubated at RT for 30 minutes. Each cell suspension was sonicated on ice for 20 seconds (pulse time 5 sec, pulse off time 5 sec, output level 2) using a Misonix™ 3000 Sonicator. The cell debris was removed by centrifugation at 16,000 x g for 10 minutes and the supernatant was assayed for protein concentration using Thermo Scientific Pierce BCA Protein Assay (Part No. 23225) or Thermo Scientific Pierce BCA Protein Assay Kit-Reducing Agent Compatible (Part No. 23252).

Sample preparation

For the Pierce protocol, HeLa cell lysate (100µg) with digestion indicator (1%, w/w) was reduced with 10mM DTT for 45 minutes at 50°C and alkylated with 50mM iodoacetamide for 20 minutes in dark at RT. Excess iodoacetamide and other contaminants were removed by acetone precipitation at -20°C for 1 hour. The protein was resuspended in digestion buffer and digested with Lys-C (1:100, enzyme:substrate) for 2 hours at 37°C followed by digestion with trypsin (1:50, enzyme:substrate) overnight at 37°C. Peptide samples were also prepared according to standard urea, FASP1, and AmBic/SDS2 methods.

LC-MS and data analysis

A Thermo Scientific EASY-nLC 1000 HPLC system and Thermo Scientific EASYSpray Source with Thermo Scientific EasySpray Column (25cm x 75μm i.d., PepMap C18) was used to separate peptides (500ng) with a 30% acetonitrile gradient in 0.1% formic acid over 100-140min at a flow rate of 300nL/min. The samples were analyzed using a Thermo Scientific Velos Pro, a Q Exactive hybrid quadrupole-Orbitrap or an Orbitrap Elite mass spectrometers. For data analysis, Thermo Scientific Proteome Discoverer software version 1.4 was used to search MS/MS spectra against the uniprot human database using SEQUEST™ search engine with a 1% false discovery rate. Static modifications included carbamidomethyl (C) and dynamic modifications included oxidation (M). The data set was screened by Preview software (Protein Metrics) for assessment of sample preparation quality. To assess the digestion efficiency, the Digestion Indicator protein sequence was included in the protein database. Five digestion indicator peptides were quantified manually with extracted ion-chromatograms of the raw LC-MS/MS data or automatically with Thermo Scientific Pinpoint 1.2 software.

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The data in this article were previously presented at the 2013 American Society for Mass Spectrometry annual meeting in a poster titled: A Versatile Sample Preparation Procedure for Shotgun Proteomic Analyses of Complex Samples by Mass Spectrometry.