Services
Services Home PageDNA Sequencer
Available Services:
Sequencing Facility services are available to the members of the Life Sciences Division at Harvard University which includes the MCB, OEB, CCB, Biological Anthropolgy, and Psychology departments. We offer the following sequencing options:
1. Staff-run analysis of dye terminator sequencing reactions.
Drop off your reactions ready to run on the sequencer. The staff will run them and put your data on the central server about 17 hours.
2. Self-run use of our 4 sequencers.
After training by our staff, you will be given “power-user” status and may run your own plates on our sequencers. Only one “power user” per lab, please.
Location:
The Sequencing Facility is located in Biolabs 3094. A small shelf next to the door is provided for submitting the samples.
Contacts:
Current Operators:
- Ben tenOever (tenOever [-at-] mcb [dot] harvard [dot] edu, Fairchild 489)
- Sean Buchanan (buchanan [-at-] mcb [dot] harvard [dot] edu, Fairchild 493 )
- Stefanie Schalm (schalm [-at-] fas [dot] harvard [dot] edu, Fairchild 481)
Any of these operators can be contacted for information regarding staff-run sequencing.
Sequencing Trainer:
- Christian Daly (cdaly [-at-] cgr [dot] harvard [dot] edu)
Contact Christian if you would like to be trained to run your own plates on the sequencer.
For questions regarding the sequencing web signup page, contact webmaster [-at-] mcb [dot] harvard [dot] edu.
For general questions about the facility, contact Claire Reardon (claire [-at-] cgr [dot] harvard [dot] edu).
Domain Accounts
All Sequencing Facility users must have an MCB or CGR Domain Account in order to use the signup and scheduling pages and to be able to retrieve sequencing data. If you don't have an MCB or CGR Domain Account, apply for one here. Requires a Harvard PIN.
Sequencing Account and Slot Sign-Up
Ask your Lab Administrator or PI for the appropriate 33-digit billing code(s); you'll need at least one to set up your Sequencing Account and to reserve sequencing slots. To add new codes to your existing account, please contact Jennifer Xie in the MCB financial office (jxie [-at-] mcb [dot] harvard [dot] edu).
Sequencing Account sign-up and slot reservations are available here. Users outside of MCB must use the log-in version in order to access the sign-up page.
The first time you use this page you will be asked to supply your name, phone number, email address and lab. Enter the correct information and click "Submit Info." This creates your Sequencing Account.
Proceed to the appropriate calendar. After you've selected a date, you will be asked for up to four 33-digit billing codes to which your run will be charged. The codes that appear in the pull-down menu will only be those for your lab (use the Code Helper to create descriptive labels for your lab's codes). After you've selected the appropriate code(s), your reservation is complete.
Sample Preparation:
Template Preparation
We recommend Qiagen miniprep kit for sequencing plasmid DNA and Qiagen PCR purification kit for sequencing PCR products.
Setting up Sequencing PCR reaction
Standard reaction contains the following:
| 8.0 uL | BigDye Terminator Ready Reaction Mix |
| 3.2 pmoles | Sequencing Primer |
| 15-30 fmoles | DNA template |
| 20uL | total reaction volume |
The standard reaction can be halved or quartered to economize the BigDye Terminator Ready Reaction Mix.
PCR Cycle Sequencing
25 cycles of
96°C 10 seconds
50°C 5 seconds
60°C 4 minutes
hold at 4°C (or store at -20°C) until ready to purify the reaction samples.
Sample Purification
Column purification of the PCR sequencing samples is strongly recommended. We recommend Centrisep columns from Princeton Separation, but there are other similar products, that should work as well.
Sample eluate from the column must be transferred to 8-strip tubes (0.2uL tubes) or plates (for example, VWR part #83009-676).
The minimal volume that can be handled by the machine is 10uL. The recommended final volume for the sample is 20uL. Therefore, we suggest addition of 10uL water to the sequencing reaction volume before column purification if your sequencing reaction was done in 10ul.
Equipment:
The facility has two 96 capillary ABI3730xl Genetic Analyzers and two 16 capillary ABI3130xl Genetic Analyzers. These instruments are configured to analyze samples produced by thermal cycle sequencing with AmpliTaq DNA Polymerase FS, and fluorescent dye-labeled dideoxy-terminators (BigDye Terminators). Detailed protocols on template preparation, PCR reaction mix preparation, cycling conditions, and post-PCR purification are available at http://www.appliedbiosystems.com. All of our instruments can also run genotyping and fragment analysis using the GeneMapper software.
A GeneAmp 2400 thermal cycler (available in Fairchild 491) is programmed with a method described by Applied Biosystems for the BigDye-terminator chemistry in a 0.2-ml MicroAmp PCR tube. Please contact the operators if cycling parameters (e.g. annealing temperature) must be different than those set in the programmed method.
Data Analysis:
MCB licenses VectorNTI for Windows and MacOS, and strongly encourages MCB department members to use it. You are welcome to use software other than VectorNTI, but if you do so, you will have to provide your own support and troubleshooting.
Instructions for downloading VectorNTI software for your PC or Mac are here (viewable only within MCB).
If you have questions about data transfer, networking, or software downloads, contact support [-at-] lsdiv [dot] harvard [dot] edu.
Core Reagents:
Following is a list of reagents mentioned in this document for your convenience.
Applied Biosystems (http://www.appliedbiosystems.com)
ABI PRISM® BigDye™ Terminator v 3.1 Ready Reaction Cycle Sequencing Kits with AmpliTaq® DNA Polymerase, FS
| 24 Ready Reactions | P/N 4337454 |
| 100 Ready Reactions | P/N 4337455 |
| 1,000 Ready Reactions | P/N 4337456 |
| 5,000 Ready Reactions | P/N 4337457 |
| 25,000 Ready Reactions | P/N 4337458 |
The Sequencing Facility sells 100-reaction aliquots for $635. To purchase an aliquot, please fill out this form and email it to 3100user [-at-] mcb [dot] harvard [dot] edu. You can pick up your aliquot from the sequencing staff.
MicroAmp® 8-Strip Reaction Tubes
| 1,000 Tubes; 125 Strips | P/N 801-0580 |
ABI PRISM™ Optical Caps, 8 Caps/Strip
| 300 strips/pkg; 2,400 caps/pkg | P/N 4323032 |
Princeton Separations (http://www.prinsep.com)
| Centrisep Spin Columns | Catalog # CS-900/CS-901 |
| Centrisep Spin Sep-8 | Catalog # CS-912 |
Staff-Run Samples
Users must have an MCB or CGR Domain Account and must register for a Sequencing Account. Please use these instructions to obtain an account if you do not already have one.
Reservations and Costs
All sample slots must be reserved on the web signup page prior to submission. Samples that haven’t been scheduled will not be loaded.
Reservations are accepted in increments of 8 samples, also referred to as a slot (1 slot = 8 samples). The facility can handle 144 slots per day. The fee per slot is $16; this will be charged to the 33-digit code that you supply when you reserve the slot. We offer a discounted price of $96 for full plates (96 samples = 12 slots). This discount will be applied automatically when you reserve 12 slots at one time.
Sample Labeling and Submission
Samples may be submitted in 0.2ml tubes or plates. Please use a plate that is compatible with the sequencer (for example, VWR part #83009-676). The samples can be capped, parafilmed or scotch taped to prevent evaporation and spills. Taped or parafilmed tubes are preferred. We do not suggest wrapping the tubes into aluminum foil as it is a problem to unwrap them. The length of exposure to light will not significantly diminish the signal.
Each submitted strip tube or plate must be clearly labeled so that the order of samples and domain account name of the user are legible. We recommend the user labeling strip tubes as indicated below using an indelible smudge-free pen, such as VWR Lab Marker. If the full name of your account cannot be written, do not worry, as we will be able to recognize user account name by looking at your user profile from the web signup. We do not need any other information - everything else is available in your user profile. Please always label the tubes and not the caps. The caps will be removed upon placement in the machine and the proper orientation can be lost.
Samples must be dropped off at the Sequencing Facility before 5pm of the loading day.
Data Retrieval
Sequence data from each sample are in a proprietary-format file containing a four-color electropherogram. The files also contain various information regarding the strength of the signal, sequence data in text format, etc. Once the samples have been analyzed, the resulting data files are transferred to the user’s folder.
Usually, the sequenced data will be put in your folder within 17 hours. This folder is only a temporary storage place – please move the data to your own permanent storage within 24 hours of receiving it, and then erase it from your folder. Data left in users' folders longer than a week may be removed without backup or warning.
Getting your files from the server:
This method can be used from a computer in Biolabs, Fairchild, or Bauer
PC:
On your desktop, right-click "My Computer"
Click on Tools, Map Network Drive
Accept whatever drive letter appears
In the folder field, type: \\titan2\dna or \\titan2.mcb.harvard.edu\dna
Scroll for your folder and click <Finish>
Your folder should open on your desktop.
Drag your files from this folder to your local drive
(it’s wise also to make a backup onto other media).
Verify that the local copies of the files are readable.
Drag the files on the server to the recycle bin and empty it.
Mac:
This only works for OS X 10.3.9 and above.
Hit Apple-K, then enter: smb://titan2.mcb.harvard.edu/dna
Scroll for your folder.
A server volume icon with Microsoft’s logo should appear on your desktop.
Double-click to open it, and drag your sequencing files to your local drive
(it’s wise also to make a backup onto other media).
Verify that the local copies of the files are readable
Drag the files on the server to the trash and empty trash.
Getting your files from the ftp site:
This method can be used from any computer with an internet connection.
You will need to use your CGR or MCB username and password to authenticate.
Point your web browser to ftp://www.mcb.harvard.edu/dna
Scroll for your folder.
Drag your files from this folder to your local computer.
(it’s wise also to make a backup onto other media).
Self-Run Samples
Each lab in Life Sciences may designate one member to become a “power user” of the Sequencing Facility. Power users are trained by our staff to independently run their plates on our sequencers. For information about our instruments, please see Equipment.
To schedule a training session, please contact Christian Daly (cdaly [-at-] cgr [dot] harvard [dot] edu). We charge a training fee of $220 per session. We will ask you for a 33-digit code that will be used to charge both the training fee and your subsequent usage fees (two different codes may be used). Please ask your PI or lab administrator for the appropriate code.
Once trained, power users are given an account that allows them to log in to the instruments. Users must also register for a Sequencing Account on the web signup page in order to be able to reserve time on the instruments. We allow reservations in one-hour time blocks. A typical run takes two hours, but longer runs may take up to four. You must also record how many plates you will run during your scheduled time so that we can correctly charge your account. You may cancel a reservation up to two hours before the scheduled start time. After that, the scheduled run will be charged to your account.
We charge a fee per plate to recover the cost of reagents and instrument maintenance; $100/plate for runs on the 3730xl sequencers or $75/plate for runs on the 3130xl sequencers. You cannot run partial plates as a power user. If you have fewer than 96 samples, consider submitting them to be run by our staff.
Once your samples have run, please come back to retrieve your data. You may transfer your data using our temporary server space on \\titan2 or with a USB key. To save to \\titan2, go to Start, Run, and type \\titan2\dna. Open your folder and transfer your data there. You can now retrieve your data from any computer with an internet connection. You will need to use your CGR or MCB username and password to authenticate. Point your web browser to ftp://www.mcb.harvard.edu/dna. Scroll for your folder. Drag your files from this folder to your local computer (it’s wise also to make a backup onto other media). Once you have confirmed the successful transfer of your data, please delete the original off the hard drive. Any data older than one week is subject to deletion without warning.
Mass Spectrometry and Proteomics
The FAS Center for Systems Biology Mass Spectrometry and Proteomics Resource Laboratory provides mass spectrometry and strategic consulting in Proteomics and Small Molecule analysis for Life Science and Chemistry researchers as well as others worldwide. This resource brings together the state-of-the-art expertise and instrumentation of the Microchemistry and Proteomics, CCB Mass Spectrometry, and Bauer Center Core laboratories, leveraging our breadth of experience to provide the best possible support for your research.
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!
Small Molecule Mass Spectrometry
The Center for System Biology Small Molecule Mass Spectrometry laboratory strives to increase the quality and availability of mass spectrometric instrumentation in the areas of accurate mass determination, confirmation and structure elucidation of organic compounds and biological samples with a wide range of mass spectrometric techniques.
Services
Two types of services are available: Open Access and Fee For Service. Open Access offers trained users a walk-up service where users can choose from various pre-programmed experimental methods and obtain results 24 hours, 7 days a week. Fee For Service allows users to submit samples with a submission form containing sample information to our facility and results are emailed to users once they are obtained. Below, please find the type of ionization methods and instruments we have in our facility for these services.
Open Access- Low resolution direct flow injection ESI MS and ESI LC/MS
- High resolution direct flow injection ESI MS
- Low resolution direct flow injection APCI MS and APCI LC/MS (available soon)
- Low resolution direct flow injection APCI/ESI MS and APCI/ESI LC/MS (available soon)
- Low resolution GC/MS
- Low resolution GC/MS/MS
- Low resolution MALDI TOF
- Low resolution direct flow injection ESI MS and ESI LC/MS
- High resoltuion direct flow injection ESI MS
- Low resolution direct flow injection APCI MS and APCI LC/MS
- Low resolution GC/MS
- Low resolution CI/EI/FAB MS
- High resolution CI/EI/FAB MS
- Low resolution MALDI TOF
*(please note the turnaround time are as follows: low resolution (~2-5 business days), high resolution (~5-10 business days). In most cases, the turnaround time is much shorter. Rush service is also available. Please call our facility for more details.
Data Analysis
Data Analysis is an essential part of mass spectrometry. Our facility provides data analysis and interpretation consultation service.
Each of the open access instrument is set up to aquire and process data automatically. In most cases, a data report is printed when acquisition has completed. Agilent LC instruments (time of flight and quadrupole) will send a data report via email and the data report can be opened using DataBrowser which can be loaded on your personal computer (Windows platform only). DataBrowser can be obtained from our facility and it will be available soon on the FAS software page.
Fee For Service results are generally delivered in pdf format wherever possible. Please feel free to contact us, before and after sample submission, and we will ensure your sample is carried out correctly. We encurage you to discuss your experimental results with us.
Forms
Two types of fee for service forms are available:
- If you have a Harvard 33 digit billing code, please download this form: Internal User
- If you do not have a Harvard 33 digit billing code, please download this form: External User
Please consult our staff if you would like to submit for GC/MS or MALDI analysis.
Rates
Open Access
| Harvard | Non-Harvard | |
| Low Resolution OA FIA | $25 | n/a |
| High Resolution OA FIA | $25 | n/a |
| Low Resolution OA ESI LC/MS | $25 | n/a |
| Low Resolution OA GC/MS | $25 | n/a |
| Low Resolution OA GC/MS/MS | $15 | n/a |
| Low Resolution OA MALDI | $45/hr | n/a |
Fee For Service
Harvard | Non-Harvard | |
| Low Resolution CI, EI or FAB | $35 | $70 |
| High Resolution CI, EI or FAB | $80 | $115 |
| Low Resolution APCI, ESI | $35 | $70 |
| High Resolution ESI | $75 | $115 |
| Low Resolution APCI or ESI LC/MS | $60 | $115 |
| Low Resolution GC/MS | $50 | $90 |
| Low Resolution MALDI | $95 | $115 |
Fees are per experiment unless otherwise noted.
Sample Preparation
Sample preapration is crucial in obtaining good results in mass spectrometry. There are several general factors namely, purity, concentration, salt content, solvent used and the nature of compound. Below, please find the guidelines to different services.
Please feel free to consult with us and we would gladly recommend the best sample preparation depending on your needs. Our contact information can be found on the Contact page.
Fee For Service
sample preparation
General Information
Six instruments are able to accept submission:
- Agilent 6120 Quadruple LC/MS with ApCI/ESI source and fraction collector
- Agilent 6210 Time-of-Flight LC/MS
- Agilent 6890 GC/MS
- JEOL SX102 magnetic sector with EI/CI/FAB methods
- Micromass LCT
- Applied Biosystems Voyager RP MALDI
Each sample MUST be submitted with a submission sheet with all fields filled out. Unless otherwise requested, all samples will be carried out by ESI before other ionization techniques will be performed. Users wishing to submit for MALDI analysis should consult the laboratory personnel before submitting samples. If you have a sensitive compound (air/moisture etc), please consult the laboratory staff to schedule an appointment
Submission Sheets
To assist our personnel optimizing experimental conditions, proposed compound structure should be provided. An internal and an external form are available. For those have a Harvard 33 digit billing code, please use the internal form. All others should use the external form. Forms can be found here: Forms
Submission Process- There is one "In" tray, located on top of the mailboxes against the wall as you enter the facility.
- Sample vials may be taped to the sheets (if stable in room temp.) or placed in the tray on the top shelf in the fridge next to the mailboxes.
- All samples MUST be labeled.
- Samples should be submitted and fully-dissolved (in suitable solvents) in chromatography auto-injector vials (available in the stockroom) at an appropriate concentration (~0.1 mg/mL).
- Partial solubility is not acceptable.
- Solid sample placed in vials may be submitted ONLY if the sample is not stable in liquid solvents and please identify a solvent that will dissolve your sample completely on the submission sheet.
Open Access
SAMPLE PREPARATION
General Information
These instruments are setup for open access:
- Agilent 1100 HPLC with fraction collector (here or new page?)
- Agilent 6120 Quadruple LC/MS with ApCI/ESI and fraction collection (coming soon)
- Agilent 6210 Time-of-Flight LC/MS
- Agilent 6890 GC/MS
- Applied Biosystems Vision Preparative Scale HPLC (here or new page?)
- Applied Biosystems Voyager DE-Pro MALDI-TOF
- Waters Quattro micro GC/MS/MS
Open Access (OA) provides a "Do It Yourself" mechanism for users to acquire mass spectra. Sample information is entered into a "Login Interface" by the user. Samples are prepared in auto injector vials and placed in the instrument's auto injector rack as instructed by the login system. Data are printed out after each sample has been analyzed. You can also log in a batch of samples. Samples need to be compatible with the instrumental solvent system and ionization technique to achieve the best results.
Submission Sheet
No submission sheet required. All user information is pre-entered and login is created.
Submission Process
Samples are prepared in solution in an auto-sampler vial. Vials must be compatible with Agilent HPLC or GC auto-samplers. Samples are dissolved in an appropriate solvent (see FAQ for compatibility issues). Obtaining a reasonable sample concentration is crucial. No more than a few ppm (parts per millions) should enter the instrument. This is because too much sample will cause a number of problems.
| Method | Mode | Concentration (mg/mL) | Concentration (ppm) |
| FIA | Positive | 0.001 | 1 |
| FIA | Negative | 0.01 | 10 |
| LC/MS | Positive | 0.01 | 10 |
| LC/MS | Negative | 0.05 | 50 |
| GC/MS | Positive | 0.05 | 50 |
Instruments
OPEN ACCESS INSTRUMENTS
Agilent 6120 Quadrupole LC/MS with Multimode source A single quadrupole instrument capable of unit mass resolution to 1500 amu. The instrument is equipped with a Multimode source capable of simultaneous APCI and ESI ionization in both positive and negative ion modes. Samples that ionize in solution are generally analyzed in ESI-only mode; those that do not are generally analyzed in APCI-only mode. Samples with poorly defined or unknown characteristics can be analyzed in Multimode combining ESI and APCI in a single run. The instrument is configured for either flow injection or HPLC introduction of samples from an automatic sampler. Samples with unique sensitivities (organometallics or air-sensitive samples, for example) can be introduced from a syringe prepared under conditions controlled by the user. The mass spectrometer is equipped with a fraction collection system allowing peak collection based on a specific mass, total ion current, UV absorbance, or a combination the three. Preparative chromatography can be accommodated at flow rates up to 5 mL/min.
Agilent 6210 Time-of-Flight LC/MS A time-of-flight instrument that is capable of unit mass resolution to 13,000 amu. This instrument is equipped with a high pressure LC binary bump and a dual nebulizer ESI source. The spectral acquisition rate can be as high as 40 spectra per second. As reported by Agilent, the mass accuracy is less than 2 ppm with an internal mass reference solution. This instrument is capable of producing both low and high resolution (to 4 decimal places) mass determinations as well as LC/MS separations with dioarray UV traces. Softwares MassHunter and EasyAccess are setup for users to walk up and login samples with few basic information entries.
Hewlett Packard (Agilent) HP6890GC/5973MSD GC/MS equipped with HP-1 30m x 0.25µm capillary column operating in the splitless mode that works for small, volatile and semi-volatile molecules. The ionization technique is electron impact (EI) and carrier gas is Helium. The acquisition software is ChemStation and an open access software is setup to allow users to choose a set of methods ranging from low to high molecular masses (50 to 800) and at different ramping rates. NIST library is also available for references.
Applied BioSystems DE Pro MALDI A linear MALDI TOF instrument with delay extraction capable of unit mass resolution to at least 1200 AMU, and lower resolution beyond that. In principle there is no upper mass limit. MALDI is generally used to characterize nonvolatile compounds (peptides and proteins, for example) and produces mainly singly charged ions in both positive and negative modes. MALDI TOF is a “gentle” ionization technique, and can be used to produce intact molecular ions when other means of analysis result in fragmentation. Samples are prepared and spotted onto a sample plate off-line, then introduced into the instrument. Data collection for each sample is rapid, usually a matter of only a few minutes.
Waters Quattro micro GC/MS/MS A triple quadrupole mass spectrometer with an Agilent 6890 GC and CTC CombiPAL autosampler has a mass range from 4 to 1500 amu. Headspace sampling allows direct volatile compounds injections. The ionization techniques are electron impact (EI) with carrier gas Helium and Chemical Impact (CI) with methane. It can be used for many methods such as multiple reaction monitoring (MRM), pproduct ion, precursor ion and neutral loss/gain. Masslynx is the acquistition software and OAlogin is the open access software.
FEE FOR SERVICE INSTRUMENTS
Applied Biosystems Voyager RP MALDI A linear MALDI TOF instrument capable of unit mass resolution to at least 1200 AMU, and lower resolution beyond that. In principle there is no upper mass limit. MALDI is generally used to characterize nonvolatile compounds (peptides and proteins, for example) and produces mainly singly charged ions in both positive and negative modes. MALDI TOF is a “gentle” ionization technique, and can be used to produce intact molecular ions when other means of analysis result in fragmentation. Samples are prepared and spotted onto a sample plate off-line, then introduced into the instrument. Data collection for each sample is rapid, usually a matter of only a few minutes.
Micromass LCT An electrospray ionization (ESI) mass spectrometer equipped with a HP1100 LC system and a syringe pump. This instrument can be used to measure low and high resolution masses in both positive and negative modes. It is suitable for small to large organic molecules ranging from 100 m/z to 3000 m/z including peptides, proteins and DNA samples. LC/MS can also be carried out using this instrument. The acquisition software is MassLynx.
Waters Q-Tof micro LC/MS/MS The Waters Q-Tof micro LC/MS/MS system consists of a time-of-flight tandem mass spectrometer equipped with both a normal and a nano ESI sources, Waters 2695 HPLC, Eksigent nanoLC and Waters Masslynx software. The instrument features two systems, normal LC/MS/MS and nano LC/MS/MS. For normal LC/MS/MS system, it has full-scan LC/MS/MS performance, the automated exact mass measurement in both MS and MS/MS modes; For the nanoLC-ESI-MS/MS system, the flow rate is from 50 to 1000 nL per minute. The great selectivity gains are observed. The sample consumption is reduced up to a few fmol for 4-mer oligonucleotide in the negative mode. The instrument is configured for either flow injection or HPLC introduction of samples from an automatic sampler. The powerful data-collection and processing tools enhance the quality and timeliness of analytical results. The Q-Tof micro is used extensively in several application areas such as, metabolite profiling, proteomics, DNA, DNA adducts and food safely as well as general analytical studies.
ADDITIONAL LC SYSTEMS
Agilent 1100 HPLC An analytical scale HPLC with a fraction collector which can be used to separate and analyze components within a chemical mixture. The injection volume of the autosampler is 1-100 uL. The pump flow rate is 0.1-2 mL/min. Chemstation software is used for control and data analysis.
Applied Biosystems Vision preparative HPLC A LC system is used for separating and analyzing large scale of chemical mixture. The AFC injection system can inject as much as 1-5 mL samples and the typical flow rate during the run is 10-20 mL/min.
Training
Open Access trainings on the following instruments are held every week. Please contact our facility to schedule a session.
Open Access Training Handout:
Agilent 1100 HPLC with fraction collector (available soon)
Agilent 6120 Quadruple LC/MS (available soon)
Applied Biosystems Vision Preparative Scale HPLC (available soon)
FAQ, Links and Tools
FAQ
Links
Tools
FAQ - Proteomics
Frequently Asked Questions
How do I best identify my protein?
Protein identification can be done either by enzymatic digestion of an in-gel sample followed by LC/MSMS analysis or direct n-terminal protein sequencing (Edman chemical sequencing). If the protein is n-terminally blocked (80% of eukaryotic proteins are naturally blocked) then digestion is the next logical step. Once digested the peptides can be separated and collected by off-line HPLC with UV detection (leading to Edman chemical sequencing of individual peptides) or the mixture analyzed on the mass spectrometers by LC/MSMS.
How much protein do I need for a successful analysis?
See the Protein Quantity Estimation section to start. Edman chemical sequencing needs 10pmol for high quality data, more for extended sequence runs. Keep in mind the initial yields for this technique is ~50%, with a repetitive yield around 92%. Typically samples can run 30 residues with ~100pmol sample loaded, depending on the protein and sample preparation. Mass spectrometry is several orders of magnitude more sensitive that Edman sequencing, thus it is possible to say we can get high quality data from as little as 10fmol protein. This amount, however, is not what was loaded on the gel, as each step in the sample handling process (reduction, alkylation, digestion, peptide extraction and concentration) incurs protein and peptide loss to surfaces. Maximizing quantity is always the first thing to do for any sample, and in some projects it is simply not possible to get more sample, so trace level analysis must be done with a subsequent loss of protein coverage and spectra quality to some degree. The degree of this impact is difficult to quantify, as each protein will behave differently and thus unpredictably at very low quantities. Also see the FAQ regarding silver stained gel spot or bands.
FAQ - Small Molecule
- Unsure what ionization technique to choose? All samples will be analyzed by Electrospray (ESI), if ESI was unable to generate satisfactory results, direct EI/CI/FAB techniques can be carried out (please check appropriate boxes on the submission sheet if you would like EI/CI/FAB analysis after ESI failed. When in doubt, please ask your friendly staff before submission. Please visit JEOL Analytical Instruments website if you would like additional information on different types of ionization methods.
- Do I need high resolution? High resolution is generally required for journal publications (≤ 1000 m/z). If you would like to have high resolution performed, on the submission sheets, please check the appropriate box for “High Res if Low Res Pass”. Please note that high resolution analysis will require a longer turnaround time and cost more than low resolution analysis.
- What sort of turn around time can I expect? If you have urgent needs, please contact us for our rush service rates. Electrospray (ESI) low resolution data usually can be obtained within 2 business days while high resolution data usually can be obtained in less than 1.5 weeks. EI/CI/FAB low resolution data usually can be obtained within 5 business days while high resolution data usually can be obtained in less than 2 weeks. Please be patient, especially when the instruments are down for maintenance or the mass spec facility is experiencing unexpected high volume submissions.
- What about MALDI analysis? Please contact us for current information.
- How do I get to use the Open Access LC/MS and GC/MS systems? Contact us for a training session. It takes about 30 minutes. No one is allowed to use the system until they have taken this course. We can also arrange for you to gain access to the lab after hours once you are trained on the Open Access instruments.
- Can I run all my samples by Open Access? The OA LC/MS instrument is equipped with an ESI ionization source. Your sample needs to be amenable to the method. While OA will provide satisfactory data in many cases, the diversity of chemistry undertaken in our department makes it impossible to give a detailed list of what will and will not work. However, the following types of materials are definitely NOT amenable: a) very volatile materials (ones you would normally submit for EI and CI). b) materials that will precipitate or decompose in polar solvents (many organometallics fit this category). c) OA GC/MS instrument is suited for semi-volatile and volatile samples with low molecular weight. Please consult with our staff for the appropriate instrument for your analysis.
- What about different solvent systems? Our solvents are Water with 0.1 % of formic acid and Acetonitrile with 0.1 % of formic acid. Samples may be dissolved in most common VOLATILE organic solvents or water. Do NOT run samples dissolved in 100% DMSO and DMF, which will damage the instrument. Please consult our staff if you have any question regarding solvent use.
- What about high resolution analysis? Our Open Access Agilent 6210 TOF LC/MS is capable of performing high resolution analysis (to 4 decimal places). Please note that for high resolution analysis, the sample should be pure.
Proteomics Useful Links
www.asms.org - American Society for Mass Spectrometry
www.abrf.org - Association of Biomolecular Resource Facilities
expasy.org - ExPASy Proteomics Server
www.hupo.org - Human Proteome Organisation's (HUPO) International Website
www.ushupo.org - Human Proteome Organisation's (HUPO) US Website
www.ionsource.com - Mass Spectrometry and Biotechnology Resource
www.ncbi.nlm.nih.gov - National Center for Biotechnology Information
www.ncbi.nlm.nih.gov/blast - NCBI BLAST search
Useful Links - Small Molecules
Personnel
Proteomics
William S Lane
Manager
John M Neveu
Staff, Senior Biomolecule Mass Spectrometrist
Bogdan A Budnik
Staff, Senior Biomolecule Mass Spectrometrist
Renee A Robinson
Staff, Research Assistant
Amir D Karger
Staff, BioInformatics and Perl Scholar
Adam K Malinowski
Staff, Applications Developer
Small Molecules
Michelle X Li
Staff, Research Scientist
Norman CY Lee, PhD
Staff, Research Scientist
Contact
Proteomics
16 Divinity Av, Biolabs BL061
Mass Spectrometry & Proteomics
Cambridge MA 02138617-495-4043 ph
617-495-1374 faxhmf [-at-] harvard [dot] edu
Small Molecule
12 Oxford Street
Mass Spectrometry Center, Bauer B-01
Cambridge MA 02138(617) 495-3571 ph
(617) 384-8382 faxmassspec [-at-] chemistry [dot] harvard [dot] edu
Sample Submission
Proteomics
- Shipping Address
Cambridge MA 02138
- Drop-off Location
Small Molecule
- Shipping Address
Harvard UniversitryMass Spec Center12 Oxford Street, Bauer B01Cambridge, MA 02138 USA
- Drop-off Location
Bauer LaboratoryRoom B01between 9AM and 5PM
Directions
Harvard Complete Campus Map: Click Here
By Car: Automobiles are not recommended since metered parking is limited. Massachusetts Avenue and Oxford Street both have some meter parking available. Please contact Harvard Parking Services for the most current parking information and availability of parking at the 52 Oxford St garage.
By Public Transportation: MBTA T (Red Line) to Harvard Square (between Central Square and Porter Exchange) and follow directions below.
From Harvard Square: Proceed North into Harvard Yard and walk towards the Science Center. Make a right at the intersection of Oxford Street and Kirkland Avenue and walk one block up Kirkland Av. Turn left onto Divinity Avenue and walk to the second driveway on your right. The Bauer Laboratory (7 Divinity Av) is on the left before the driveway, the Biological Laboratories (16 Divinity Av) is ahead on the right just after the driveway.
Research Computing
Please refer to our new location on the FAS IT website:
> Life Sciences Research Computing
Consulting
Please refer to our new location on the FAS IT website:
Infrastructure
We manage a number of systems and services to make researchers more productive. Some of our systems and ways we can help are outlined below:
- Cluster (number of nodes, OS, LSF/queues)
- Desktop (AD, A.V., SMS)
- Email and Calendaring (POP, IMAP, HTTP)
- Software
- Storage
- User Accounts
- Web Hosting (disk, OS/IIS, Apache)
Cluster
The Life Sciences cluster consists of the following hardware:
14 nodes: Dual Intel® Xeon™ CPU 3.06GHz, 2GB RAM, RHEL3 U8 ×86
36 nodes: Dual Intel® Xeon™ CPU 2.80GHz, 4GB RAM, RHEL3 U8 ×86
14 nodes: Dual Intel® Xeon™ CPU 2.80GHz, 2GB RAM, RHEL3 U8 ×86
22 nodes: Dual Intel® Xeon™ CPU 2.80GHz, 2.5GB RAM, RHEL3 U8 ×86_64
12 nodes: Quad Intel® Xeon™ CPU 2.80GHz, 8GB RAM, RHEL4 U3 ×86_64
1 node: Quad Genuine Intel® CPU 3.00GHz, 8GB RAM, RHEL4 U4 ×86_64
1 node: Dual Core AMD Opteron™ Processor 275, 4GB RAM, x86_64
1 node: Intel® Xeon™ CPU 3.20GHz, 4GB RAM RHEL4 U4, x86_64
1 node: Dual Core Intel® Xeon™ CPU 2.40GHz, 1GB RAM, RHEL4 U4, x86
Total: 229 cpus
Cluster head node: portal.cgr.harvard.edu
Dual Intel® Xeon™ CPU 3.06GHz, 2GB RAM, RHEL3 U8 ×86
Cluster Network: 1Us with 1GB NIC, IBM Blades chassis with 4 trunked ports, DellBlades with 1GB pass-through all connected into a Foundry Networks FastIron 1500 switch. 10 DellBlades have Infiniband network as well as 1GB pass-through network.
Admin host: serving the cluster is a IBM Blade server running DHCP, LSF licenses, Mathematica licenses. It contains Dual Intel® Xeon™ CPU 3.00GHz, 4GB RAM, RHEL4 U4 ×86
Disk Storage: All Nodes mount a central EMC SAN (through NSXs) on the cluster network via NFS. Each node has local scratch space which varies from 13GB to 67GB.
Tape Backup Robot: ADIC Scalar i2000 - 600 tape unit.
Backup Infrastructure: EMC Networker v7.2.2 on Linux, NDMP clients are 6 EMC NSXs. All zoned into one Cisco Fiber switch. Sustained tape backup speeds of 110-140+ MB/s from one NSX to one LTO-3 tape drive.
Queuing System: LSF v6.0 from Platform Computing (www.platform.com)
Current queues defined:
QUEUE_NAME PRIO STATUS MAX JL/U JL/P JL/H NJOBS PEND RUN SUSP
interact 86 Open:Active - 1 - - 0 0 0 0
dellblades 85 Open:Active - - - - 460 416 44 0
delllong 84 Open:Active - - - - 4 0 4 0
rsoft 83 Open:Active - 2 - - 0 0 0 0
hunter 82 Open:Active - - - - 0 0 0 0
giribet 81 Open:Active - - - - 80 0 80 0
flybase 70 Open:Active - - - - 0 0 0 0
blades 65 Open:Active - - - - 0 0 0 0
CGRshort 60 Open:Active - 50 - - 0 0 0 0
CGRnormal 50 Open:Active 40 - - - 460 420 40 0
CGRlong 40 Open:Active - 60 - - 0 0 0 0
short 30 Open:Active - 20 - - 0 0 0 0
normal 20 Open:Active - - - - 5285 5161 123 1
long 15 Open:Active - 10 - - 24 0 24 0
interact - for interactive jobs with a MAX of 12hrs runtime, 1 job per
user dellblades - for jobs to run on Dellblades1-10, primary group that uses these is the karplus group.
rsoft - for jobs from the Lieber lab to run rsoft based jobs, only run on host cfa15 for 12hrs MAX runtime.
hunter - for jobs from the hunter lab group to run on hunter1, they have priority access to this machine.
giribet - for jobs from the Giribet lab to run on giribet1-2. They have
exclusive access to these machines.
flybase - for jobs from the Flybase group, to run in hosts fb1-22. They have priority access to fb1-22.
CGRshort - for jobs from CGR members that have a MAX runtime of 1hr.
CGRnormal - for jobs form CGR members that have a MAX runtime of 24hrs. maximum of 40 jobs total at any one time.
CGRlong - for jobs from CGR members, no time limit. Maximum of 60 jobs per users at any one time.
short - for all external or CGR members that run jobs for a MAX of 1hr runtime. Total of 20 jobs per user.
normal - for all external or CGR members that run jobs for a MAX of 24hrs runtime.
long - for all external or CGR members, no time limit, 10 jobs MAX per user.
Desktop
:::Last Updated on 13NOV2006:::
Each new desktop assigned to a new Life Sciences individual will be an up-to-date desktop system with the following features:
- Pentium-4 @ > 3.2Ghz
- > 1.0gb RAM
- > 200gb Hard Drive
- > 17" LCD Display
The current desktops being issued are the Dell Optiplex GX620 and more information about them can be found here.
Additionally all Windows computers will come issued with the following software (in its most up-to-date version):
- Symantec Antivirus Corporate or McAfee Antivirus
- Microsoft Office with frontpage
- Adobe Acrobat Reader
Lastly, the desktop will be part of the domain and be subject to both antiviurs and Windows updates at regular intervals.