Biomolecule Mass Spectrometry & Proteomics
Proteomics is the study of the proteins expressed in a given system over its life, at specific time points or under varying conditions, which has closely followed recent advances in mass spectrometry and bioinformatics. The Mass Spectrometry and Proteomics Resource Laboratory was established to assist researchers in existing and new methods of protein characterization and quantitation in the context of systems biology.
We strongly advise discussing your project with us before sample preparation.
hmf [-at-] harvard [dot] edu
Services
Protein Identification
Protein identification can be done either by direct n-terminal chemical sequencing or by digestion and LC/MS/MSMS analysis. After an in-depth project discussion, the sample is prepared by the user following simple protocols, and submitted to the facility for analysis. Samples are enzymatically digested, run on nano-capillary HPLC/MSMS, and the MSMS spectra are correlated against a specific database for peptide identification. When applicable, N-terminal Edman sequencing is available.
Complex Mixture Analysis
Complex mixtures of proteins are identified by a number of single- and multi-dimensional approaches. For example, GeLC, in which an entire lane of an SDS-PAGE gel is excised into sections, affords the user a two dimensional separation of the protein mixture based on protein intact molecular weight (SDS-PAGE) and then individual peptide hydrophobicity by reversed phase chromatography (RPLC). A similar method known as MUDPit (Multidimensional Protein Identification Technology) starts with a solution digestion of the sample, then two dimensional chromatography by strong cation exchange chromatography (SCX) followed by reversed phase chromatography (RPLC).
Posttranslational Modification Site Determination
Starting with a single highly purified protein in an SDS-PAGE gel slice, multiple sites of modification, eg. phosphorylation, acetylation and others, can be determined. This process involves a detailed project discussion and careful selection of multiple enzymes to maximize peptide coverage for specific sites of interest.
N-terminal Edman Sequence Analysis
N-terminal sequence analysis is a chemical method in which the amino terminal amino acid is labeled with phenylisothiocyanate and specifically cleaved, followed by identification of the released phenylthiohydantoin amino acid by RPLC. This process can sequence upwards of 30 amino acids given sufficient quantities, typically 10pmol or more, of a single protein. Edman sequencing affords the researcher the ability to characterize the N-terminus of a protein directly, including quantitatively differentiating between even single amino acid cleavage sites. This process gives true de-novo sequence information, as it is not a database dependent technique, and has been an established method in protein research for many decades.
C-terminal Sequence Analysis
In this lab, we use multiple enzymes to obtain redundant peptides which exhaustivly define the C-terminal region of a purified protein. Multiple instrument runs are combined with custom bioinformatics tools to provide the final result.
De novo Sequence Analysis
All of the previously mentioned mass spectrometry techniques rely on the protein sequence being known and available for comparison of mass spectra to a database. If this is not the case, identical peptides from homologous proteins can often be found, leaving many still unidentified. While software algorithms have advanced, these spectra often require expert manual interpretation. This laboratory has over 40 years of combined experience specializing in de novo interpretation.
Quantitative Proteomics
One of the major challenges in modern proteomics is characterizing the differences in protein expression between two or more samples in a statistically relevant method. For instance, these methods could show differences in protein expression between treated and non-treated cell lines, healthy and sick animals, or between knockout and wild type organisms.
Labeled: Quantitative mass spectrometry normally utilizes stable isotope labeling at the whole cell level, intact protein level or even peptide level. There are several well established techniques to do this, and a detailed project consultation prior to beginning an experiment with this goal is mandatory.
- SILAC (stable isotope labeling with amino acids in cell culture) is a simple and straightforward approach for in vivo incorporation of a label into proteins for mass spectrometry (MS)-based quantitative proteomics. SILAC relies on metabolic incorporation of a given ‘‘light’’ or ‘‘heavy’’ form of the amino acid into the proteins. The method relies on the incorporation of amino acids with substituted stable isotopic nuclei (e.g. deuterium, 13C, 15N). Thus in an experiment, two cell populations are grown in culture media that are identical except that one of them contains a ‘light’ and the other a ‘heavy’ form of a particular amino acid (e.g. 12C and 13C labeled L-lysine, respectively). When the labeled analog of an amino acid is supplied to cells in culture instead of the natural amino acid, it is incorporated into all newly synthesized proteins. After a number of cell divisions, each instance of this particular amino acid will be replaced by its isotope labeled analog. Since there is hardly any chemical difference between the labeled amino acid and the natural amino acid isotopes, the cells behave exactly like the control cell population grown in the presence of normal amino acid. It is efficient and reproducible as the incorporation of the isotope label is 100%, however is generally applicable only to cell or tissue culture experiments due in large part to the expense of stable isotope labeled growth media.
- ICAT (Isotope Coded Affinity Tags) is a well publicized method of relative quantification in which two or more different samples are labeled at the intact protein level with isotope coded tags. The samples are combined for digestion, and the labeled peptides are specifically removed by affinity chromatography to one part of the label (biotin-avidin enrichment). These labeled peptides can then be run together, with isotopically different peptides eluting near each other at a mass difference of a couple of amu in the full MS scan for quantitative comparison of expression levels between samples. A newer version has acid labile labels, which has improved the chromatographic performance of the process. This process has the advantage of labeling intact proteins prior to digestion, which can reduce system variability.
- iTRAQ (Isotope Tags for Relative and Absolute Quantitation) is another popular technique that includes up to 10 isotopic labels for multiplexing experimental variables. The technique is based upon chemically tagging the N-terminus of peptides generated from protein. The labeled samples are then combined (post labeling), fractionated by nano-LC and analyzed by tandem mass spectrometry. Peptides are chromatographically resolved as single peaks with identical full MS masses. Fragmentation of the labeled peptides generates a low molecular mass reporter ion that is unique to the tag used to label each of the samples. Measurement of the intensity of these reporter ions, enables relative quantification of the peptides in each digest and hence the proteins from where they originate. This process has the advantage of no chromatographic interference from the labels but requires a low mass MSMS scan to observe the reporter ions.
- AQUA – method of absolute quantitation based on synthetic "heavy" peptides that is used as an absolute standard. A fundamental goal of cell biology is to define the absolute levels of every protein expressed by an organism under the conditions of interest. Precise measurement of protein changes in terms of molecules per cell, and for all expressed components, would provide the high quality datasets necessary for a comprehensive understanding of disease at the molecular level. Absolute quantification of proteins uses 13C- and 15N- labeled synthetic reference peptides and tandem MS to measure expression in terms of number of molecules per cell. This process targets specific peptides individually and can become expensive but provides the most exact quantitation in many cases. Typically a SIM or SRM experiment is preformed on the mass spectrometer to target only the peptides of interest, so the method can be adapted to high throughput proteomics experiments.
Label-free methods for quantitation have recently become popular and shown good results in blind studies that have been published. These processes rely on highly reproducible chromatography; typically with high pressure sub-2 micron particle reverse phase columns and traps, to produce statistically relevant data. The Q-TOF premier is one system that targets this type of analysis directly, with the Protein Expression System and nano-Acquity UPLC. This system eliminates the isolation step of MSMS data acquisition, relying on post-run analysis to construct individual MSMS spectra from the mix of MSMS data.
Intact Molecular Weight Determination by MALDI-TOF
Intact proteins, oligonulceotides and peptides frequently need an intact mass determination, and MALDI-TOF is the prefered method to obtain this information due to the "soft" ionization technique and low charge states associated with this technique. Samples are applied to a target with an approproate matrix and allowed to dry fully, concentrating of sample in a crystalized matrix spot. A UV laser imparts energy to the sample through the matrix, causig the sample to ionize (typically a a singly or doubly charged species) and the time it takes to travel along the flight tube is proportional to the mass of the sample molecule. Typically proteins and peptides between 0.5 and 200kDa, and oligonucleotides up to 10kDa can be observed at very high sensitivity. Sample concentration is key to good signal quality, and salts, detergents and other compounds in the sample buffer can reduce the ionization of the molecule significantly.
Intact Protein Molecular Weight Determination by ESI
Molecular weight determination of intact protein can be done by direct Electro Spray Ionization (ESI) on the Q-TOF Premier. A concentrated solution of desalted protein is sprayed into the instrument, with a series multiple charge state envelopes the expected result. This data is then deconvoluted to intact protein mass measurement, typically up to 200kDa, depending on the homogeniety of the sample.
Amino Acid Analysis
Amino acid analysis is another time-proven technique of protein characterization for composition and quantitation. A protein sample is fully hydrolyzed with vapor phase HCl, the free amino acids derrivitized with PITC, then quantitated by RPLC as compared to an amino acid standard. Samples should be salt, amine and detergent free in a highly concentrated form. Alternativly, samples can be prepared from an SDS-PAGE gel and electroblotted to PVDF following the directions for Edman chemical protein sequencing.
Forms
Sample Submission
- Mass Spec (Protein ID, PTM, C-term)
Use this form for submitting samples in gel or in solution for internal sequencing, modification site determination (PO4, acetylation, ubiquination), C-terminal sequencing, crosslinking modification / add mass project. - Mass Spec (Intact MW determination)
Use this form for submitting samples that require an intact protein mass determination (MALDI or ESI). - N-terminal Sequencing
Use this form for submitting samples for N-terminal Edman sequencing. - Amino Acid Analysis
Use this form for submitting a sample that requires composition or quantitation from a solution or PVDF membrane.
Miscellaneous
- Add-Weight Calculation
Use this form if you are submitting a sample for modification site determination using a reagent or other biomarker with a known add mass for which we will need the exact molecular weight. - "Email Your Sequence" Instructions
Use this form if you are submitting a sample for a modification project on a known protein.
Rates
Note bene: We strongly recommend a thorough discussion of your project before sample preparation.
Service | Harvard | Academic | For Profit |
Enzymatic Digestion | 90 | 125 | 150 |
HPLC or LCMS (instrument run only) | 95 | 125 | 150 |
Protein ID Analysis and Report | 60 | 130 | 150 |
Additional Analysis (PTMs, C-term, etc.) | 50 | 90 | 110 |
Consultation (first hour) | 60 | 130 | 150 |
Consultation (additional hours) | 50 | 90 | 110 |
Edman N-terminal Sequencing (5 cyc min.) | 200 | 300 | 400 |
Edman Sequencing (cycles 6 and up) | 25 | 40 | 50 |
Amino Acid Analysis | 50 | 70 | 90 |
MALDI-TOF of proteins or peptides | 45 | 70 | 90 |
MALDI-TOF additional work (per 30 min) | 45 | 70 | 90 |
Zip tip prep for MALDI-TOF | 45 | 70 | 90 |
Direct ESI -MS analysis (per 30 min) | 95 | 125 | 150 |
Instrumentation
ThermoFisher LTQ-Orbitrap
The ThermoFisher LTQ-Orbitrap combines the best of linear ion trap technology in the LTQ, with the novel Orbitrap mass analyzer, which provides high mass accuracy and resolution for both MS and MSn spectra. This instrument is typically run with the Orbitrap acquiring full MS data at low ppm mass accuracy and resolution up to 100,000 HWFM, while the LTQ acquires multiple MSMS scans of peptides for identification and modification site determination. Other operational configurations include SRM/MRM experiments, high energy collisions in the C-trap, and high accuracy MSMS spectra in the Orbitrap. This instrument is essential and continually amazes us with the sensitivity and accuracy we achieve on a daily basis.
Driving the chromatography on the Orbitrap is the Waters nanoACQUITY UPLC system. This system maintains accurate gradient production in a splitless configuration in the sub-μl range at pressures up to 10,000psi. The autosampler allows automated trapping for sample concentration and desalting and provides excellent reproducibility and linearity across the injection volume range of 0.1 to 20μl.
New Objective Inc. provides the nanocapillary chromatography hardware and ESI source used exclusively on the LTQ-Orbitrap. The PicoView source allows easy positioning and visualization of the ESI spray, which is key to operation of the high sensitivity nanocapillary columns with integrated spray tips (Picofrit columns) and trap columns (IntegraFrit columns) provided by this long-time collaborator with our lab. No other chromatographic configuration has demonstrated the versatility, robustness and extremely high sensitivity that these products provide.
Waters Q-TOF Premier
The Waters Q-TOF Premier is a hybrid quadrupole TOF instrument equipped with the Protein Expression System for non-labeled quantitative proteomics analysis. The instrument features Lock Mass spray and MSe, which allows this instrument to skip the isolation step for MSMS, greatly increasing the effective duty cycle when run in this mode. Key to the expression analysis is the stability and reproducibility of the nanoACQUITY UPLC system and the high pressure 1.7μ reversed phase capillary column, which afford the instrument excellent chromatographic properties. This instrument is also capable of intact protein mass determinations by direct ESI infusion and has the full suit of data analysis software from Waters.
Applied Biosystems DE-STR MALDI-TOF Matrix Assisted Laser Desorption Time Of Flight (MALDI-TOF) is s soft ionization technique capable of analyzing proteins, peptides, oligonucleotides and other organic molecules intact up to a mass range of 200 kDa. This instrument provides a complimentary method of screening samples prior to more complicated ESI mass determinations, and can be set up to screen many samples in an automated fashion.
http://www.appliedbiosystems.com/
Applied Biosystems Procise 494-HT
Edman sequencing is a classic protein characterization technique that has been a mainstay in this laboratory for over 20 years. This process requires >1pmol of protein, either in solution of on PVDF blot, and can quantitatively determine the sequence of one or several protein or peptides with a cycle time (one amino acid per cycle) of about 45 minutes. This is not a database dependent technique, so it can be considered the original form of denovo protein sequencing. Typical sequences are 10 amino acids, but extended sequence runs can be preformed for novel proteins or peptides.
Amino Acid Analysis & RPLC/SCX Chromatography on Agilent 1100
One Agilent 1100 HPLC is configured for automated Amino Acid Analysis (AAA) for protein quantitation and composition. A second 1100 system can perform Strong Cation Exchange (SCX) and Reverse Phase Liquid Chromatography (RPLC) with automated peak detection and fraction collection for sample prefractionation and purification under various configurations including diode array detection (DAD) and variable wavelength detection (VWD). Controlled by Agilent ChemStation software, these systems provide flexibility and robustness for protein and peptide work.
Project Consultation
One of the hallmarks of this facility is the attention we pay to project discussion, sample preparation, and final data presentation for our clients. Bill Lane, John Neveu, and Bogdan Budnik are the scientists who conduct these discussions, ensuring that your experimental design and samples have been prepared in an optimal fashion for their intended analyses.
Please call 617-495-4043 and leave a detailed message including your contact information and brief project description, or alternativly send an e-mail to hmf [-at-] harvard [dot] edu including the same information to be placed into our calling queue. We strictly adhere to our telephone queue, with the earliest contact called first, so be patient and we will call you back in turn.
Sample Preparation
Sample preparation is integral to getting the most from the resources available at this facility. The links below cover some of the basics in sample preparation for various services; however it is very strongly recommended that researchers discuss their projects with a staff scientist well in advance of sample preparation.
- Protein Quantity Estimation
- Edman Chemical Sequencing (and Amino Acid Analysis on PVDF)
- In-Gel Proteolytic Digestion for LC/MSMS Protein Identification
Sample Quantity Estimation
Estimating Protein Quantity
An excellent method of estimating the quantity of protein is based on density in a 1x8mm band on an SDS-PAGE gel.
A sharp 1 x 8 mm band on a gel (0.75mm) holds on average 1 μg when saturated and will be a very dark Coomasie Blue stain. Be conservative and avoid convincing yourself that volume of gel contains more. You scale your estimate relative to this by both intensity and area (volume).
1. Evaluate your intensity on a scale of 1 to 5 where 5 is the darkest Coomassie band you observed in your career, 1 is a very faint, near threshold Coomassie, and 3 is an average stain.
2. Estimate the gel area in mm2
3. Calculate the micrograms of protein as follows
μg protein = ( intensity / 5 ) x ( area / 8)
4. Once you have an estimation of mass and molecular weight, calculate the picomoles of protein present
picomole protein = 1000 x μg protein / M.W. (in kDa)
Thus, if you have a very darkly stained 1 x 8 mm band (~1ug), for a 25 kDa protein, you have 40pmol present . You would, however, have only 10pmol for a 100 kDa protein, and only 4pmol for a 250 kDa protein.
Do not rely on comparison to MW markers as a method since by doing so you are normalizing back to what was present in the tube, not what is available in the gel for digestion.
Edman Chemical Sequencing
Sample preparation for Edman sequencing
The protocol guidelines below are not a substitute for a discussion of your project. Always call to discuss the specifics of your project before submitting a sample. Successful projects stem from a thorough understanding by both the researcher and our laboratory of goals, expectations and requirements.
- Edman sequencing requires 10pmol or more protein for high quality extended sequence runs. (See Protein Quantity Estimation). Maximize quantity of sample you can commit to this experiment.
- Once a certain quantity has been achieved, density is the second most important factor, as the reaction cartridge is only 9mm wide and can hold only a few 1x8mm strips of PVDF before the flow is impeded in the instrument. Maximize density of the final gel bands.
- Reduce background proteins with fresh preparations of all gel based buffers, stains and wash solutions. Segregated or new glassware and gloves should be used for all steps in the process.
- Once you have maximized the quantity of protein and have found the optimal SDS-PAGE conditions for maximum density of the final protein bands, run the sample under these conditions.
- Electroblot the sample to PVDF membrane, by whatever protocol you are most comfortable with.
- Stain the PVDF with Ponceau red. Amido black is a second choice stain (more aggressive), leaving coomassie blue as a poor choice for PVDF, but it can still be used with care.
- Wash the blot to remove excess stain. Generally the same solution as the stain without the coloring agent is fine to remove excess stain.
- Cut out the bands of interest tightly, leaving no unstained PVDF. Place then into a 1.5ml clear plastic snap-top micro Eppendorf tube.
- Rewet the PVDF with 1drop methanol, then wash the blots with 1ml reagent grade water and vigorous vortexing for 1 minute. Repeat this wash twice. Ø Remove all liquid and let blot air dry.
- Label tube(s), ship over packed (in a small box or other container) to prevent breakage or crushing of the sample tube. There is no need to ship on dry ice as long as the blots are dry.
- Include the Protein Sequence Analysis sample form, filled out completely, along with the sample. A hardcopy of the PO# you will be using to pay for the analysis should also be included.
- If this is a recombinant protein, please include the expected sequence, which will be used to align the final result into a more meaningful report if the data is heterogeneous.
- A minimum of 5 cycles can be run, with a maximum of ~30 amino acids expected for most cases.
- Solution samples can also be submitted; however salts and other buffer components must be eliminated for quality sequence data. Normally a detailed discussion will be needed in these cases.
Preparation for In Gel Digestion
Preparation for In Gel Proteolytic Digestion and High Sensitivity Technologies
The protocol guidelines below are not a substitute for a discussion of your project. Always call to discuss the specifics of your project before submitting a sample. Successful projects stem from a thorough understanding by both the researcher and our laboratory of goals, expectations and requirements.
· Commit as much protein as is possible
Regardless of how sensitive our technologies are, always prep as much protein as possible. DO NOT SKIMP. If it only takes a week to generate a "one-X prep" then spend two weeks and prep 2X. All sequencing technologies, MS or Edman, are mole-based not mass based: e.g. for equal staining a 200K protein has 10X less than a 20K, of course.
Density (protein to gel volume ratio) matters. There is no restriction on the number of lanes that you send, as long as you have saturated and focused your protein in each lane. Large quantities of protein in diffuse gel volumes can fail; similarly, the way to make small amounts of protein succeed at state-of-the-art levels is to do everything you can to focus that amount in the least gel volume. (Also note this in excising your bands below.)
Run a standard gel that optimizes for the above conditions. Always using the highest quality and fresh reagents/solvents throughout all of these procedures. Reducing, non-reducing, native or gradient conditions are OK as long as the amount of protein has been maximized and the amount of gel minimized. Please contact us if special or unusual conditions or reagents are used and note these on your form.
There is evidence that overall recovery maximizes in a gel of 1 mm thickness. Gels of 0.5mm thickness can have greater loss of protein during the stain and destain processes.
Please remember that the only function of stain in this experiment is to observe your band. Therefore only stain long enough to accomplish this goal. If your protein requires five hours to visualize then so be it. However, do not stain for 5 hours if your protein is visualized in 15 min. General recipes can be used--contact us only if you need to use a very non-standard condition or reagent.
Coomassie and other stains are interfering artifacts in our technologies. Destain long enough to clear your background and still have nicely visualized bands. All excess stain should be removed.
To maximize the protein to gel volume ratio, excise only the hearts of the bands. Do not include any unstained gel. For example, adding an extra 0.5mm of gel around a 1 x 10mm average band doubles its area, yet the amount of protein gained may be < 5%.
One tube per protein, even if there are multiple lanes or spots. DO NOT SPLIT protein bands into multiple tubes. Individual tubes are considered as different samples and will be processed and charged separately.
Use plain tubes. There are three items to avoid: 1) no colored tubes, 2) no O-rings and 3) no chemical treatment.
This should be from the same gel, processed identically to your samples. This is a blank gel to control for chemical noise and generalized, nonspecific protein background. We only need one control for each 3 to 4 proteins that are submitted.
Each wash consists of 0.5 - 1.0 ml of 50% HPLC grade acetonitrile/water for 2 - 3 min. with gentle shaking, discarding the supernatant after each wash. This step is to normalize your gel slices for storage and shipment.
Do your best to remove all excess liquid. A drop or two is fine. The gel slices remain moist.
Parafilm can introduce chemical noise into the system. Be careful of introducing dust and surface material to the cap or lip of tube.
Any temperature -20 °C or below is fine. Samples have been successfully processed a year later. For that reason, always identically prepare other bands from the same gel at the same time, even if you are not planning to send them. This will allow you to revisit them if they become significant at a later time.
If your shipment is international, it is your responsibility to clear all customs issues before sending. Our address is on the forms.
The form is your lifeline to each sample. Do not leave anything blank. We encourage you to briefly outline your goals and the biochemical significance in the instruction area. If there is information you have that is relevant to interpretation of the analysis or handling of your sample, let us know on the form! The Digestion/Separation form is the only form to fill out, but one is required for each tube, including control. Do NOT fill out a Protein Sequence form or Mass Spectrometry form.
High sensitivity analysis comes with a price: keratin contamination is always observed. Wearing gloves is not enough. In fact, the typical contamination is believed to be not your own hands but rather non-specific dust contamination being introduced in the steps after one has run the gel. Observed keratins do not necessarily have to be at the same MW as your band. Note: you cannot get rid of it but you can minimize it. Rinse all surfaces while you work, (simple rinses with HPLC grade water), that contact the gel from the point that you take the gel apart to the final storage. These surfaces include: the outside of your gloves, staining trays, scalpels, razor blades, tongs and the inside of the final 1.5ml tubes. Note bene: the less protein you have the more significant the non-specific keratin background will be to successful analysis of your sample. Regardless what organism you are working with, do not be surprised when keratin is observed by us.
Login ("Day 0"): Note: most delays are due to incomplete forms and billing information.Week 1 (day 7-10): In gel reduction, alkylation and proteolytic digest of 100% of the sample(s).Week 2 (day 10 -18): High sensitivity LC/MS of ~10% of the digest mixture. Purpose: Ascertain whether digest was successful. A fax will be sent that simply indicates whether we are proceeding or not. There is no detailed data analysis at this point and usually no need for a discussion.Week 3 (day 15 - 25): In depth review of acquired MS/MS spectra before consuming the remaining 90% of the digest. MS/MS sequencing by MS/MS correlation analysis and reporting.Week 4 (day 20 - 30): Discussion of project results. Further HPLC analyses and single sequence attempts, if necessary.
Colloidal Coomassie, copper, zinc and modified silver stains are compatible with the downstream technologies with significant caveats. Please discuss with us before using. Reading the literature is not a substitute for a thorough discussion with us. Remember the obvious: a more sensitive stain does not give you more protein!